Method, probe and kit for DNA in situ hybridization and use thereof

ABSTRACT

The invention relates to a method for the detection of the occurrence of initiation of replication events in genomic DNA in a eukaryotic cell, involving contacting said eukaryotic cell comprising said genomic DNA with a first nucleotide probe, under conditions enabling in situ hybridization of said first nucleotide probe with a target region in the DNA genome, wherein said target region comprises a nucleic acid sequence which has no identified corresponding annealing RNA in a metabolically active cell and therefore remains RNA-free during transcription and replication of said DNA genome and detecting said first nucleotide probe hybridized to said DNA. Further detection of at least one RNA molecule can be achieved. The invention also relates to a nucleic acid molecule suitable for use as a probe, hybridizing with a target region in a eukaryotic genomic DNA, and comprising a nucleic acid sequence which has no identified corresponding annealing RNA in the metabolically active cell containing said eukaryotic genomic DNA and therefore remains RNA-free during transcription and replication of said DNA genome. The invention also encompasses kit(s) for carrying out in situ hybridization and use of the method(s), nucleic acid molecule(s) or kit(s) of the invention in the detection of mitochondrial disease(s), neoplasic diseases(s) or cancer(s), or in the testing of the cytotoxicity of organic or chemical compounds, especially drugs, on eukaryotic cells.

The invention is directed to a method for analyzing events associatedwith replication in the genomic DNA in a eukaryotic cell, especially amammalian cell, using in situ hybridization techniques. The method ofthe invention is especially directed to the detection of the occurrenceof initiation of replication events in genomic DNA. In a particularembodiment, the invention also enables the co-detection of DNA and RNAmolecules in a single cell. According to a further particularembodiment, the invention enables the co-detection and co-visualisationof DNA and optionally RNA and protein(s) in a single cell.

In the context of the invention, a genomic DNA molecule is a nucleicacid molecule that belongs to the genome of a cell, and replicates,especially in an autonomous or independent manner, in particular underthe control of cellular regulatory elements, in metabolically activecell(s), and whose replication can therefore be observed in situ. Thegenome of a cell is considered to be the DNA of an organism, thatcarries all the information for all the proteins the organism will eversynthesize, and more generally the genome contains all the informationnecessary for the survival of a cell. Genomic DNA can be chromosomal DNAor plasmidic DNA, with the proviso that said plasmidic DNA belongs tothe genome of a cell, as defined herein. More particularly in thecontext of the invention, genomic DNA (gDNA) molecule(s) is eithermitochondrial gDNA or nuclear gDNA or both. A plasmid is a DNA moleculethat is separate from, and can replicate independently of, thechromosomal DNA. Plasmids are double stranded and, in many cases, inparticular in most cases, circular. Plasmids usually occur naturally inbacteria, but are sometimes found in eukaryotic organisms. By contrast,plasmidic DNA not belonging to a genome as functionally defined is notconsidered to be genomic DNA in the context of the present invention.

The invention also relates to specific probes, in particular nucleotideprobes, which are particularly devised for the detection of theoccurrence of initiation of replication events in genomic DNA in aeukaryotic cell. The invention encompasses means useful for detectingthe occurrence of initiation of replication events in genomic DNA, inparticular kits comprising such probes and processes for carrying outthe invention.

According to particular embodiments, the methods, probes and kits of theinvention are suitable for analyzing initiation of replication ofgenomic DNA at the single cell level, and therefore provide means fordetecting impaired replication of gDNA, and in particular means usefulfor detecting diseases associated with such impairment, includingmitochondrial disease(s), neoplasic diseases(s) or cancer(s).

In a particular embodiment, the invention especially relies on theresults obtained in experiments designed to observe the occurrence ofinitiation of replication events in mitochondrial genomic DNA in humancells.

Indeed, mitochondrial DNA (mtDNA) replication and transcription arecrucial for cell function, but these processes are poorly understood atthe single-cell level. Tools currently offered to biologists do notpermit the specific detection of mitochondria engaged in initiation ofDNA replication. With respect to nuclear gDNA, tools currently offeredto biologists do not permit the detection of nuclear gDNA engaged ininitiation of its replication within a cell, i.e. at the single celllevel, not isolated from its cellular context.

Mitochondria are ATP-producing organelles whose function is directed notonly by the nuclear genome but also by their own genome. Eachmitochondrion carries several copies of a genomic DNA, i.e. a circulardouble-stranded DNA that is replicated and transcribed autonomously inthe organelle. Mitochondrial DNA (mtDNA) is arranged in nucleoproteincomplexes, nucleoids, that include factors involved in replication andtranscription as well as structural proteins required for mitochondrialmaintenance (Chen and Butow 2005; Spelbrink 2010). These proteinsinclude DNA polymerase γ (Polγ, the enzyme responsible for replicationof mtDNA, and TFAM (also known as mtTFA), a protein implicated both intranscription of and in binding to the mtDNA, and whose levels arecorrelated with those of mtDNA (Poulton et al. 1994; Falkenberg et al.2007; Shutt et al. 2010). Human mtDNA, a 16.5 kbp molecule, is organizedin 13 protein-coding, 2 rRNA, and 22 tRNAs genes that are transcribedfrom the (heavy) H-strand (12 mRNA, 2 rRNA and 14 tRNAs) and from the(light) L-strand (1 mRNA for ND6 gene, and 8 tRNAs) with production ofpolycistronic precursor RNAs. These primary transcripts are processed toproduce the individual mRNA, rRNA and tRNA molecules¹. The prevalentview of mtDNA replication is that DNA synthesis starts from origin O_(H)where the nascent H strand frequently terminates 700 bp downstreamgiving rise to the 7S DNA, which produces a characteristic triplestranded structure, the D-loop^(2,3). When leading strand synthesis hasreached two thirds of the genome, it exposes another major origin, theorigin of L-strand DNA replication (O_(L)), and lagging-strand DNAsynthesis then initiates in the opposite direction. Conversely, coupledleading and lagging strand synthesis has been described in a reducednumber of molecules⁴, suggesting that this model is not fullyelucidated. Mitochondria display a variety of shapes ranging from highlyinterconnected tubular structures to individual small spherical units.These structures are highly dynamic and can be regulated bymitochondrial fusion and fission, and they vary during cell growth (Chan2006; Lee et al. 2007; Mitra et al. 2009). Whether these differentstructures are related to mtDNA processing needs clarification.

Nuclear DNA (nDNA) is generally compacted in chromosome(s), and itsreplication generally begins at specific location(s) in the genome,called “origin(s)” or “replication origin(s)”, which is/are thepositions at which the DNA helix is first opened, giving rise to a“replication bubble”. Unwinding of DNA at the origin, and synthesis ofnew strands, forms a replication fork, which has an asymmetricstructure. The DNA daughter strand that is synthesized continuously isknown as the leading strand, whose synthesis slightly precedes thesynthesis of the daughter strand that is synthesized discontinuously,known as the lagging strand. Eukaryotic chromosomes generally containmultiple origins of replication. The different replication origins ineukaryotic chromosomes can be activated in a sequence, determined inpart by the structure of the chromatin, with the most condensed regionsof chromatin beginning their replication last.

The processing of mitochondrial DNA has been intensively analysed withbiochemical approaches (reviewed in^(5,6)), which essentially examinedglobal cellular and mitochondrial populations, but little is known aboutmitochondrial activity at the single cell level, or about DNA and RNA atthe single cell level. Thus, several questions on the dynamics and theregulation of mtDNA transcription and replication inside the same cellremain unresolved, as well as their implication in cellular fonction. Todate, studies on mtDNA replication are widely based on molecular biology(2D-Gel of replication intermediates and in vitro assays). Currentlyavailable fluorescence in situ hybridization (FISH) tools, includingrecent improvements⁷, do not identify mitochondria engaged in DNAreplication, and they do not discriminate the transcription profiles oforganelles in single cells. If a technique allowing to detect onemitochondrial transcript at a time has been disclosed³⁰, it required agenetically engineered step and was shown for the transcript ND6 only.

Moreover, although sequential RNA and DNA labelling⁸, as well aslabelling of either RNA or DNA, and proteins^(9,10) have been performed,the techniques used, namely immunofluorescence and Fluorescent In SituHybridization (FISH), did not permit to simultaneously detect proteinsand mitochondrial DNA and RNA (triple detection). Consequently,available techniques do not render possible a powerful and directlyexploitable observation of the course of events occurring during gDNA,especially mtDNA, transcription and replication, especially whendifferent molecular subpopulations, such as DNA, RNA or even proteins,are involved.

Therefore, the present invention addresses the need to obtain anoutstanding tool for studying DNA replication, allowing a deepercomprehension of the events associated with DNA replication, andincluding the comprehension of the coordination of these events withother events such as RNA transcription and even protein(s) distributionin cell(s) or tissue(s) by tracking and monitoring these distinctmolecular subpopulations in a concomitant or even simultaneous manner.Consequently, the invention proposes a new way to further explore thecomplex cellular dynamics and to use such exploration in detection ofpathological states.

According to a particular embodiment of the present invention novelinformation can be provided on the dynamics of gDNA, especiallymitochondrial gDNA, processing during physiological and pathologicalprocesses. These findings have implications in diagnostic tools ofdiseases, especially mitochondrial diseases or diseases associated withmitochondrial dysfunction(s) or impairment, in particular those wheremtDNA depletion and mtDNA loss can be observed.

Indeed, defects in the mitochondrial replication machinery can lead toloss of genetic information by deletion and/or depletion of themitochondrial (mt) DNA, which subsequently may cause disturbed oxidativephosphorylation and neuromuscular symptoms in patients. mtDNA depletioncan originate from genetic defects, or be acquired, i.e by clinicaltreatments, as for prolonged administration of anti-HIV nucleosideanalogues. qPCR analysis on mtDNA is currently used to detectalterations of the mtDNA content in a given cell population. Bypermitting the monitoring of the occurrence of initiation of replicationevents in mitochondrial genomic DNA, and for example by measuring theseevents and following their evolution during the progression of disease,the present invention provides for the determination of such an impairedor abolished function. Consequently, detecting the initiation of DNAreplication events and optionally combining this detection with thedetection of other signals, therefore determining the state of mtDNA atthe single cell level, enables the emergence of a more powerful researchand diagnostic tool.

To this end, the invention relates to a method for the detection of theoccurrence of initiation of replication events in genomic DNA in aeukaryotic cell, comprising the steps of:

-   -   contacting said eukaryotic cell comprising said genomic DNA with        a first nucleotide probe under conditions enabling in situ        hybridization of said first nucleotide probe with a target        region in the DNA genome, wherein said target region comprises a        nucleic acid sequence which has no identified corresponding        annealing RNA in a metabolically active cell and therefore        remains RNA-free during transcription and replication of said        DNA genome and,    -   detecting said first nucleotide probe hybridized to said DNA.

According to a particular embodiment, the target region or the nucleicacid sequence of the target region that has an ability to remainRNA-free, is located in a naturally transiently open structure of twocomplementary single strands of gDNA in a metabolically active cell.

The methods and uses according to the invention are performed in vitroon samples of biological material.

According to a particular embodiment, the expression “the target regionin the DNA genome that comprises a nucleic acid sequence which has noidentified corresponding annealing RNA in a metabolically active cellremains RNA-free during transcription and replication of said DNAgenome” encompasses the case wherein said nucleic acid sequence remainssubstantially RNA-free during transcription and replication of said DNAgenome, meaning that the amounts of RNA transcripts that would be foundannealed to said nucleic acid sequence remain below the detection levelin experimental conditions of detection of hybridization according tothe invention, in particular they do not influence the experimentsconducted herein, i.e. their signals do not exceed background levels ornoise.

Especially, it is indicated that the amounts of such transcripts wouldnot be sufficient to allow their detection after treatment with DNAse,in particular DNAseI according to the invention. These amounts wouldrepresent minor amounts with respect to the whole amount of transcripts.

In the context of the invention, a “target region” in the DNA genome isa genomic DNA region comprising a nucleic acid sequence which has noidentified corresponding annealing RNA in the metabolically active cellunder assay and therefore remains RNA-free during transcription andreplication of the DNA genome to which the nucleic acid sequencebelongs.

In other words, a target region is a genomic DNA region comprising asequence domain that has ability to remain RNA free, in particularRNA-transcript(s) free, during the transcription and replication of theDNA genome to which it belongs (mtDNA or nDNA) in the cell that istested.

According to a specific embodiment, the target region of said firstprobe consists of a nucleic acid region in a genomic DNA which has noidentified corresponding annealing RNA in a metabolically active cell.

In a particular embodiment, the target region of said first probeencompasses said RNA-free domain but is longer than said domain,including substantially longer, as illustrated hereafter.

According to a particular embodiment, such a naturally transiently openstructure can be a replication bubble originating around the locus of areplication origin. According to a particular embodiment, such anaturally transiently open structure is the so-called DNA encompassed bythe D-loop region of the mitochondrial genome, which is located betweencoordinates 16024 to 576 in the human mitochondrial genome (NCBI orGenbank or MITOMAP sequence reference NC_012920.1). According to aparticular embodiment, the sequence of the target region that has anability to remain RNA-free, is located proximal to, or includes, oroverlaps known origin(s) of replication, in particular a mitochondrialorigin of replication, or one of its ends is within a distance of lessthan 10 nucleotides, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9, especially6 nucleotides, from a known origin of replication, in particular amitochondrial origin of replication. In particular in human mtDNA, suchan origin of replication can be the O_(H) origin of replication, locatedbetween coordinates 110 to 441 of the mitochondrial genome (NCBI orGenbank or MITOMAP sequence reference NC_012920.1).

According to a particular embodiment said target region or said nucleicacid sequence of the target region encompasses nucleotides within adistance of less than 10, in particular 1, 2, 3, 4, 5, 6, 7, 8 or 9nucleotides upstream or downstream from a naturally transiently openstructure of two complementary single strands of gDNA in a metabolicallyactive cell.

In the context of the invention, “hybridization” relates to the fact ofobtaining a close interaction of the nucleotide probe and the targetregion that is expected to be revealed by the detection of thenucleotide probe. Such an interaction can be achieved by the formationof hydrogen bonds between the nucleotide probe and the target sequence,which is typical of the interactions between complementary nucleotidemolecules capable of base pairing. Hydrogen bonds can be found, forexample, in the annealing of two complementary strands of DNA.

Within the context of the invention, hybridization conditions encompasscontacting the nucleotide probe with the target region during about 15hours at 37° C. in a conventional buffer such as illustrated in theexamples, e.g. an hybridization buffer with 50% formamide, 10% dextransulphate, in 2×SSC pH 7.0 or another similar buffer as appropriate.Hybridization conditions can further involve a step of washing the cellcontacted with the nucleotide probe(s) with an appropriate,conventional, buffer prior to the detection step, such as illustrated inthe examples.

For example, washing can be performed several times for 2-5 min in 2×SSCbuffer at room temperature, then several times in 1×SSC buffer at roomtemperature, then several times in 0.1×SSC buffer at room temperature.Finally, an ultimate washing can be performed several times in PBS 1×buffer at room temperature.

The nucleic acid sequence of the probe should be at least partlycomplementary to the sequence of the target region of the genomic DNA,i.e. should be complementary over a region sufficient to enable stablebase pairing.

Typically, a first nucleotide probe designed for hybridizing to a targetregion of genomic DNA is a labelled nucleic sequence fragmentcomplementary to the target region of the genomic DNA and havingsubstantially or in particular exactly, the same length as said target.

In a particular embodiment, the first nucleotide probe designed forhybridizing to a target region of genomic DNA is a labelled nucleicsequence fragment complementary to the targeted DNA fragment and havingsubstantially, or in particular exactly, the same length as the nucleicacid sequence which has no identified corresponding annealing RNA in ametabolically active cell and therefore remains RNA-free duringtranscription and replication of said DNA genome.

In a particular embodiment, a first nucleotide probe designed forhybridizing to a target region of genomic DNA is a labelled nucleicsequence fragment comprising a nucleic acid sequence that iscomplementary to the targeted DNA fragment, said nucleic acid sequencehaving substantially, or in particular exactly, the same length than thenucleic acid sequence which has no identified corresponding annealingRNA in a metabolically active cell and therefore remains RNA-free duringtranscription and replication of said DNA genome.

However, according to other embodiments, the interaction of thenucleotide probe and the target region can also involve van der Waalsinteractions, ionic bonds or covalent linkages. Such interaction(s)might imply that the nucleotide probe contains modified nucleotides orbear specific moieties generally not present in nucleotidic molecules.

“In situ hybridization” refers to the fact that the hybridization iscarried out on the assayed biological material. Said biological materialcan be single cell(s) or tissue(s), or a sample comprising the same.Preferably, the integrity of the structure and/or content of thebiological material is maintained. Therefore, in order to achieve theinvention, the biological material is preferably fixed.

Accordingly, in a preferred embodiment, the method of the invention iscarried out on fixed cell(s) or tissue(s).

In a particular embodiment, the method of the invention further permitsto maintain the integrity of the cell(s) volume and thus the analysis offixed sample(s) in three-dimension.

According to a particular embodiment, the cell(s) or tissue(s) areeukaryotic cell(s) or tissue(s), in particular human cell(s) ortissue(s). For illustration cell(s) or tissue(s) derived from human celllines such as HeLa, HCT116, HT29, AGS cell lines, and/or human primarycells, i.e. IMR-90, BJ human fibroblasts obtained from ATCC, are used.

In a particular embodiment, the genomic DNA is mitochondrial gDNA.

In another particular embodiment, the genomic DNA is nuclear gDNA.

In another particular embodiment, the genomic DNA is both nuclear gDNAand mitochondrial gDNA. In such an embodiment, the expression “genomicDNA” refers to a group of genomic DNA molecules, i.e. refers to morethan one genomic DNA molecule, said group of genomic DNA moleculesconsisting of more than one copy of genomic DNA molecules (as found in asingle mitochondrion) and/or more than one genomic DNA molecules thatare different from each other (such as mitochondrial gDNA and nucleargDNA).

According to the invention, a “probe” is aimed at revealing the targetregion of interest, and is therefore generally, but non-exclusively,labelled. Labelling of the probe aimed at revealing the target region inthe DNA genome is preferably achieved with either radio- orantibody-discoverable- or fluorescent- or biotinylated-tags or quantumdots, especially fluorescent quantum dots. Said tags or quantum dots aredirectly or indirectly associated, including coupled, to the probe.Depending upon the type of labelling, the probe can be localized orvisualized or measured on the biological material after hybridizationwith its target using appropriate techniques, such as autoradiography orfluorescence microscopy. An example of discoverable tag is digoxigenin,biotin, or hapten for example revealed by a labelled antibody or alabelled reagent, such as a fluorescent antibody raised againstdigoxigenin or a labelled biotin binding molecule such as avidin orstreptavidin.

According to a specific embodiment, the probe is rendered discoverable,especially through fluorescence detection methods, by introducing anantigen in said probe or by coupling said probe with an antigen thatwill be further revealed by a secondary anti-antigen antibody,especially a fluorescent anti-antigen antibody. One advantage of usingantibodies might be an increase of the intensity of the resultingfluorescent signal.

In a particular embodiment, probe(s) are directly labelled withfluorescent moieties (tags). One advantage of such an embodiment mightbe to bypass the use of an antibody for the detection of the probe inorder, for example, to increase the specificity or the practicability ofthe labelling/detection method.

Probe(s) is/are preferably nucleotide probe(s), and are especially shortsequences of single stranded DNA capable of base pairing with theircomplementary DNAs. The invention encompasses probe(s) containingnucleotide(s) coupled or linked to other molecule(s) or moiety(ies).

According to a particular embodiment probe(s) is/are DNA probe(s) suchas PCR product(s) or DNA fragment(s), including plasmidic probe(s) orprobe(s) comprising such elements.

According to a particular embodiment, they can be double-stranded DNAprobes that require being denaturated as single stands prior to theiruse.

According to a particular embodiment, the nucleotide probe contains,among its nucleotides, one or more modified nucleotides or nucleotidesbearing specific moieties generally not present in nucleotidicmolecules. Locked Nucleic Acids (LNA) are modified nucleotides and aclass of RNA analogs that have an exceptionally high affinity towardscomplementary DNA and RNA. They can substitute natural nucleotides inDNA probes.

In a particular embodiment of the invention, the term “probe”encompasses more than one molecular entities used together to reveal thetarget region.

In such a particular embodiment, the target region can be fragmentedalong the considered genomic DNA. For example, a target region of theDNA genome can be spread among more than one location on a singlegenomic DNA molecule or on more than one genomic molecule.

According to a particular embodiment, the method of the invention ischaracterised in that the first nucleotide probe consists of at leasttwo subsets of probes, wherein at least one subset hybridizes with atarget region in the DNA genome that comprises a nucleic acid sequencewhich has no identified corresponding annealing RNA in a metabolicallyactive cell and therefore remains RNA-free during transcription andreplication of said DNA genome.

In a specific embodiment, one of said subsets of probes contains nucleicacid molecules comprising, or consisting of, or being fragments of, orhaving at least 80% identity with, SEQ ID NO:17.

According to another embodiment the first probe of the invention islonger than the sequence complementary to the RNA-free domain andespecially comprises a sequence hybridizing to mtDNA that is transcribedin a metabolically active cell.

By “occurrence” it is meant that the method of the invention enables toqualitatively detect initiation of the replication of genomic DNA and,according to a particular embodiment, to quantitatively detect suchinitiation event(s) of the replication process.

“Initiation of replication events” can be, for example, the formation ofreplication bubble(s) on the analyzed genomic DNA, or in a particularembodiment where the mitochondrial gDNA is assayed for initiation ofreplication, the formation of a D-loop structure, including theformation of three-stranded D-loop structure. Such events may precedethe entire replication of the analyzed gDNA, meaning that such eventsmay precede replication over the complete analyzed gDNA. Such events mayalternatively be followed by interrupted synthesis of the nascent strandof DNA.

In a cell, DNA replication usually begins at specific location(s) in thegenome, called “origin(s)” or “replication origin(s)”. Once polymeraseshave opened the double stranded genomic DNA molecule, an area known as a“replication bubble” forms (usually initiated at a certain set ofnucleotides, the origin of replication).

With respect to particular embodiments aimed at detecting the occurrenceof initiation of replication events in mitochondrial genomic DNA, it isknowledgeable to consider that, in Mammalian, Avian, Fish, or Plantcells, the initiation of mitochondrial genome replication generallyoccurs in a particular region named “control region” or “D-Loop” or“displacement loop”. When the mtDNA initiation of replication starts,the D-Loop region is opened, and the corresponding DNA locally resultsin single strands that serve as template for the synthesis of new mtDNA.This transitory opened D-Loop region may present a triplex-DNAstructure, said structure being however located in a naturallytransiently open structure of two complementary single strands of gDNA.The presence of D-loop region is typical of the human and other mt DNAs,such as Mammalian, Avian, Fish or Plant mtDNAs. The coordinates of saidD-loop regions vary according to the considered organisms but can befound in the literature³¹. However D-Loop regions are not found in allmtDNA.

In particular in human cells, the D-loop region is roughly locatedbetween the coordinates 16024 and 576 of the L-strand on themitochondrial genome (according to the data released to date ondatabases, in particular under accession number NC_012920.1 (NCBI,GenBank or MITOMAP sequence reference), see in particular MITOMAP:http://www.mitomap.org/MITOMAP/HumanMitoSeq).

In a particular embodiment, the target region or the RNA-free nucleicacid sequence comprised in said target region is located in a naturallytransiently open structure of two complementary single strands of gDNAin a metabolically active cell, as disclosed above and in the followingembodiments.

According to a particular embodiment, the target region or the RNA-freenucleic acid sequence comprised in said target region is locatedupstream from the major H-strand promoter on the mitochondrial genome(PH1), which coordinates are given in Table 2 (coordinates and directionare given herein with respect to the L-strand of the mtDNA). However, inthe context of the invention, the target region or the RNA-free nucleicacid sequence comprised in said target region encompasses the sequencesfound on either the L-strand or the H strand at the specific locationmentioned herein. Reference is made to the L-strand to indicate theposition of the major H-strand promoter only.

According to a particular embodiment, especially when probe(s) aresynthesized or obtained as a result of a PCR amplification, probe(s)is/are double-stranded DNA probe(s). They are denaturated as singlestands prior to their use. When used simultaneously after denaturation,such a mix of complementary single-stranded probe(s) results inannealing both the L and H strands of the target region of a mtDNA.

In a particular embodiment, the target region or the RNA-free nucleicacid sequence comprised in said target region is located downstream fromthe L-strand promoter on the mitochondrial genome (LP or LSP), whichcoordinates are given in Table 2 (coordinates and direction are givenherein with respect to the L-strand of the mtDNA). However, in thecontext of the invention, the target region or the RNA-free nucleic acidsequence comprised in said target region encompasses the sequences foundon either the L-strand or the H strand at the specific locationmentioned herein. Reference is made to the L-strand to indicate theposition of the L-strand promoter only.

By “naturally transiently open structure of two complementary singlestrands of gDNA” it is meant a gDNA structure formed by the dissociationof the two DNA strands constituting the gDNA as a result of processingby replication machinery and mechanism(s) inherent to a metabolicallyactive cell, during its life cycle.

More specifically, it will be understood that the target region islocated near or in a region of the genomic DNA that is involved in theearly events of the replication of said genomic DNA, such as a regionfound in a replication bubble or a region at least partly encompassed bya replication bubble. Such a region will generally be localized in thevicinity of a replication origin of a genomic DNA of an eukaryotic cell,in particular in the close vicinity or near, i.e. no farther than 10nucleotides from a replication origin of a genomic DNA of an eukaryoticcell.

According to a particular embodiment, the target region is located inthe vicinity of a replication bubble or at a locus encompassed by areplication bubble (where a replication bubble can be found), inparticular no farther than 10 nucleotides from such a bubble or locusencompassed by such a bubble. Replication bubbles initiate at the locusof replication origins.

According to a specific embodiment, the target region is located at 5nucleotides of the O_(H) replication origin in the human mtDNA.

A nucleic acid sequence having “no identified corresponding annealingRNA in a metabolically active cell” is a sequence having no strictlycorresponding, i.e. complementary or matching, RNA, especially no RNAtranscript(s) resulting from the transcription process occurringnaturally in the living eukaryotic cell under assay in the detectionconditions disclosed herein. By “corresponding” it is understood asubstantial, in particular a strict, complementarity of nucleic acidsequences which are aligned and whose similarity is calculated over theentire length of the aligned sequence by alignment algorithm such as theNeedelman and Wunsch algorithm (a substantial similarity or perfectmatch is expected). Therefore such a nucleic acid sequence has anability to remain RNA-free, in particular RNA-transcript(s) free, withinthe analyzed cell, meaning that such a nucleic acid sequence will notgive rise to any identified RNA molecule, especially a RNA molecule thatwould have been transcribed from genomic DNA in the analyzed cell, norhybridize with RNA primers involved in replication process in ametabolically active cell containing said nucleic acid sequence. Such anucleic acid sequence cannot be detected by a probe aimed at detectingthe result of transcription events occurring in a cell.

However, the fact that the RNA-free nucleic acid sequence is a sequencehas no strictly corresponding, i.e. complementary or matching, RNA,especially no RNA transcript(s) resulting from the transcription processoccurring naturally in the living eukaryotic cell under assay, does notprevent the target sequence of the first probe from having a particularportion which is complementary to RNA transcript(s) or a portionthereof. According to a particular embodiment, a nucleic acid sequencehaving “no identified corresponding annealing RNA in a metabolicallyactive cell” is therefore a sequence having no identified correspondingannealing RNA within a specific portion of its sequence, in particular asmall portion of its sequence, e.g. in a portion representing less than70% or less than 50% or less than 20% or less than about 10% of thetarget.

According to a particular embodiment, a nucleic acid sequence having “noidentified corresponding annealing RNA in a metabolically active cell”is a sequence having substantially no strictly corresponding, i.e.complementary or matching, RNA, especially no RNA transcript(s)resulting from the transcription process occurring naturally in theliving eukaryotic cell under assay in the detection conditions of thetranscripts disclosed herein. Such a nucleic acid sequence remainssubstantially RNA-free during transcription and replication of said DNAgenome, meaning that the amounts of RNA transcripts that would be foundannealed to said nucleic acid sequence remain below the detection levelin experimental conditions of standard detection of hybridization, inparticular they do not influence the experiments conducted herein, i.e.their signals do not exceed background levels or noise.

Especially, it is indicated that the amounts of such transcripts wouldnot be sufficient to allow their detection after treatment with DNAse,in particular DNAseI according to the invention. These amounts wouldrepresent minor amounts with respect to the whole amount of transcripts.

It is pointed out that such a sequence may be identified starting fromthe literature describing transcription and replication processes ofgDNA or in databases, having regard to annotation(s) available in saiddatabases, or as a result of deductions arising from said annotations.

According to a particular embodiment, such a nucleic acid sequencehaving “no identified corresponding annealing RNA in a metabolicallyactive cell” or “substantially no identified corresponding annealing RNAin a metabolically active cell” is a sequence located near orencompassing an origin of replication of a genome. The particularlocalization of said sequence, i.e. located proximal to, or including,or overlapping known origin(s) of replication(s), is as described above.

Consequently, in a particular embodiment, a first nucleotide probe whosesequence would strictly match the sequence of a nucleic acid sequence asdiscussed above, would not hybridize with any RNA molecule naturallyexpressed within a metabolically active cell.

Such a nucleic acid sequence is thus characterized in that it does notbear any coding information that would be reflected at the transcriptionlevel of the DNA processing in a cell.

According to a particular embodiment, a first nucleotide probe whosesequence would substantially match the sequence of a RNA free nucleicacid sequence as discussed above, or at least match said sequence over asubstantial portion of its whole length, would substantially nothybridize with any RNA molecule naturally expressed within ametabolically active cell, or would not hybridize on the whole lengthwith such RNA molecule.

According to a particular embodiment, the target region referred toherein is located proximal to the O_(H) replication origin in the humanmtDNA, and in particular comprises, encompasses or consists of the DNAsegment of mitochondrial gDNA localized between nucleotide position 446and nucleotide position 162024 on the H strand of the mt genome, whichcorresponds to sequence SEQ ID NO: 19, of the mitochondrial genome of ahuman eukaryotic cell, said segment extending over a length of about 80to about 1200 nucleotides. According to a particular embodiment, thetarget region referred to comprises, encompasses or consists of the DNAsegment of mitochondrial gDNA localized between nucleotide position 446and nucleotide position 16366. According to a particular embodiment, thetarget region referred to herein is located proximal to the O_(H)replication origin in the human mtDNA, and in particular comprises,encompasses or consists of the DNA segment of mitochondrial gDNAlocalized between nucleotide position 544 and nucleotide position 162024on the H strand of the mt genome, which corresponds to sequence SEQ IDNO: 1 concatenated with SEQ ID NO: 19, of the mitochondrial genome of ahuman eukaryotic cell, said segment extending over a length of about 80to about 1200 nucleotides. By definition, nucleotide positions areindicated with respect to the H strand.

SEQ ID NO: 19 is defined as nucleotide sequence: ttctttc atggggaagcagatttgggt accacccaag tattgactca cccatcaaca accgctatgt atttcgtacattactgccag ccaccatgaa tattgtacgg taccataaat acttgaccac ctgtagtacataaaaaccca atccacatca aaaccccctc cccatgctta caagcaagta cagcaatcaaccctcaacta tcacacatca actgcaactc caaagccacc cctcacccac taggataccaacaaacctac ccacccttaa cagtacatag tacataaagc catttaccgt acatagcacattacagtcaa atcccttctc gtccccatgg atgacccccc tcagataggg gtcccttgaccaccatcctc cgtgaaatca atatcccgca caagagtgct actctcctcg ctccgggcccataacacttg ggggtagcta aagtgaactg tatccgacat ctggttccta cttcagggtcataaagccta aatagcccac acgttcccct taaataagac atcacgatg gatcacaggtctatcaccct attaaccact cacgggagct ctccatgcat ttggtatttt cgtctggggggtatgcacgc gatagcattg cgagacgctg gagccggagc accctatgtc gcagtatctgtctttgattc ctgcctcatc ctattattta tcgcacctac gttcaatatt acaggcgaacatacttacta aagtgtgtta attaattaat gcttgtagga cataataata acaattgaatgtctgcacag ccactttcca cacagacatc ataacaaaaa atttccacca aaccccccctcccccgcttc tggccacagc acttaaacac atctctgcca aaccccaaaa acaaagaaccctaacaccag cctaaccaga tttcaaattt tatcttttgg cggtatgcac ttttaacagtcaccccccaa ctaac.

SEQ ID NO:19 corresponds to the human mt DNA fragment betweencoordinates 16024 and 445 of the circular human mt gDNA, resulting fromthe concatenation of the fragment between positions 16024 and 16568 andthe fragment between positions 1 and 445 (numerotation is made on theH-strand), from Genbank sequence reference NC_012920.1.

By “conditions enabling in situ hybridization” it is meant that thetarget region is rendered physically accessible to the probe in order toenable the hybridization of said probe to the target region.

According to a particular embodiment, the hybridization of the probe tothe target region may be only partial along the entire length of theprobe or the target region, but sufficient to be specific and stableduring washing step(s) following the hybridization.

According to another embodiment, the hybridization of the firstnucleotide probe to the target region occurs over the length of theprobe and/or over the length of the target region. Conditions for saidhybridization are defined above.

According to a particular embodiment, to render the target regionaccessible to the probe in order to enable the hybridization of saidprobe to the target region, said target region has to be available underthe form of an accessible single stranded of gDNA even transientlyduring the replication process. According to a particular embodiment, itis the nucleic acid sequence which has no identified correspondingannealing RNA that has to be available under the form of an accessiblesingle stranded of gDNA even transiently during the replication process.

According to a particular embodiment, the first nucleotide probestrictly anneals to the above-mentioned nucleic acid sequence comprisedin the target region that has no identified corresponding annealing RNAin a metabolically active cell. In other words, in said embodiment, whenhybridizing to the nucleic acid sequence comprised in the target regionwhich has no identified corresponding annealing RNA, the first probedoes not overflow the boundaries of said nucleic acid sequence.

Considering the mitochondrial genomic DNA, the D-loop region of said DNAcan be found as a specific structure involving a three-stranded DNAstructure that is formed when a newly synthesized single DNA strandremains bound to one of the parental DNA strand of the gDNA anddisplaces one of the duplex parental strand.

When present, such a three-stranded DNA structure might help rendering atarget region located in the D-loop structure or in the vicinity of thisstructure accessible to the probe. The target region is, in thisconfiguration, either located in a naturally transiently open segment oftwo complementary single strands of gDNA when the mitochondrial genomicDNA is entering replication, or in a region which is impacted by thepresence of the third DNA strand that might help to push aside proteinsor other elements that might render the target region crowded and/orhinder the target region with the result of rendering said regioninaccessible to the probe for subsequent hybridization of said probe tothe target region.

In the context of the invention however, initation of replication isconsidered an event to be detected in mitochondrial gDNA even when thepresence of a third DNA strand in a D-loop does not further give rise tothe replication of the whole mitochondrial DNA strand. In other words,the initiation of the replication of the mitochondrial gDNA isconsidered to happen with the formation of a replication bubble,including the formation of a D-loop around the locus of a replicationorigin.

In a specific embodiment, when the method of the invention is applied tothe detection of the occurrence of initiation of replication events inmitochondrial genomic DNA, the method of the invention can permit thespecific detection of the D-loop region opening by labeling themitochondrial genomic DNA with a probe according to the inventionhybridizing at least partly the target region located in the vicinity ofsaid D-loop or at a locus included in said D-loop.

According to a particular embodiment, the first nucleotide probestrictly anneals to the above-mentioned nucleic acid sequence comprisedin the target region that has no identified corresponding annealing RNAin a metabolically active cell.

According to a particular embodiment, the accessibility of the targetregion to the first probe of the invention can be improved by performinga step aimed at partially denaturing the genomic DNA molecule comprisingthe target region, for example by heating the eukaryotic cell comprisingsaid genomic DNA at a temperature in the range of 72 to 78° C.,preferably 75° C., for 2 to 8 minutes, preferably 4 to 5 minutes, inparticular 5 minutes, prior to the hybridization step.

According to a particular embodiment, said partial denaturation isperformed without using any chemical agent resulting in a completedenaturation of nucleic acids. Consequently, treatments with HCl orPepsin, alkaline agents or ethanol are prohibited. Conversely, the useof chemical agents and/or temperature conditions enabling or assisting apartial denaturation of nucleic acids is possible. An example ofchemical agent that can be used is formamide. Combinations between theproposed treatments disclosed herein are encompassed by the presentinvention. By “partial denaturation” it is meant that the two strandsconstituting a double stranded nucleic acid are not found completelyseparated i.e. under the form of single strands, after such adenaturation. According to a specific embodiment, said partialdenaturation results in increasing the size of opening(s) or bubble(s)that could be found on the double-stranded nucleic acid of gDNA prior toeliciting its partial denaturation.

According to a particular embodiment wherein temperature and/or chemicalagent(s) is(are) used to assist the partial denaturation of thedouble-stranded target nucleic acid, said agent(s) enable(s) the partialdenaturation by performing or assisting the increase in size ofopening(s) or bubble(s) on the double-stranded target nucleic acid, tothe exclusion of the result consisting in the dissociation of thestrands of the double-stranded target nucleic acid, on their wholelength. Accordingly, partial denaturation is a denaturation step whichleads to relaxed single-stranded DNA in a double-stranded DNA molecule.

In the context of the invention, it might be advantageous to track boththe initiation of genomic DNA replication in a cell or tissue andproduced RNA molecules, being for example RNA molecules corresponding totranscription products of genomic DNA fragments concomitantlytranscribed in said cell or tissue.

Thus, in a particular embodiment, the method of the invention is usedfor the further detection of at least one RNA molecule corresponding toa transcribed region of a DNA molecule in an eukaryotic cell, whichcomprises the step of contacting said eukaryotic cell expressing saidRNA molecule with at least a second nucleotide probe, and detecting saidsecond nucleotide probe after hybridization with said RNA molecule.

According to a particular embodiment, the DNA molecule giving rise tothe RNA transcript molecule detected by the second nucleotide probe is agenomic DNA inside said cell (the analyzed cell).

In a particular embodiment, the labelling of the nucleic acid sequenceof the target region on the DNA genome and the RNA molecule is achievedin one step, in particular simultaneously.

In a particular embodiment, the detection of the nucleic acid sequenceof the target region on the DNA genome and the RNA molecule is achievedin one step, in particular simultaneously.

In the context of the invention, the hybridization of the secondnucleotide probe to a RNA molecule corresponding to a transcribed regionof a DNA molecule is achieved by obtaining a close interaction of thenucleotide probe and the RNA molecule that is expected to be revealed bythe detection of the nucleotide probe. Such an interaction can beachieved by the formation of hydrogen bonds between the nucleotide probeand RNA molecule, which is a typical example of the interactions betweencomplementary nucleotide molecules. Hydrogen bonds can be found, forexample, in the annealing of two complementary strands of DNA.

Typically, a nucleotide probe designed for hybridizing to a RNA fragment(RNA molecule) is a labelled nucleic sequence fragment complementary tothe RNA fragment to detect. The nucleic acid sequence of the probeshould be at least partly complementary to at least a part of the RNAmolecule to detect, i.e. should be complementary over a regionsufficient to enable stable base pairing.

However, according to other embodiments, the interaction of thenucleotide probe and the RNA molecule can also involve van der Waalsinteractions, ionic bonds or covalent linkages. Such interaction(s)might imply that the nucleotide probe contains modified nucleotides orbear specific moieties generally not present in nucleotidic molecules.

In a particular embodiment, probe(s) are directly labelled withfluorescent moieties (tags).

Probe(s) is/are preferably nucleotide probe(s), and are especially shortsequences of DNA or RNA (cRNA probes or riboprobes) that binds to theircomplementary RNAs. The invention encompasses probe(s) containingnucleotide(s) coupled or linked to other molecule(s) or moiety(ies).

According to a particular embodiment, the nucleotide probe containsmodified nucleotides or bears specific moieties generally not present innucleotidic molecules. Locked Nucleic Acids (LNA) are modifiednucleotides and a class of RNA analogs that have an exceptionally highaffinity towards complementary DNA and RNA. They can substitute naturalnucleotides in DNA or RNA probes.

The invention encompasses the use of probes suitable for revealingseveral distinct RNA molecules or fragments thereof, or the use of asingle probe targeting distinct RNA molecules or fragments thereof(specific of a pool of RNA molecules or fragments thereof), or the useof a single probe specific of the sequence of a unique RNA molecule orfragment thereof within a cell or tissue. In this context the term“probe” encompasses a plurality of molecular entities used together toreveal one or many RNA molecules, said RNA molecules being distinct ordifferent.

According to a particular embodiment, DNA or RNA molecules targeted byeither a first nucleotide probe or a second nucleotide probe may beinvolved in a RNA/DNA structure.

According to the invention, a probe suitable for revealing RNAmolecule(s) is generally, but non-exclusively, labelled. Labelling ofthe probe can be achieved with either radio- or antibody-discoverable-or fluorescent- or biotinylated-tags or quantum dots, especiallyfluorescent quantum dots. Said tags or quantum dots are directly orindirectly associated, including coupled, to the probe. According to thetype of labelling, the probe can be localized in the biological materialusing appropriate techniques, such as autoradiography or fluorescencemicroscopy, respectively. The developments made above with respect tothe labelling of the probe aimed at revealing the target region in theDNA genome are also applicable to the labelling of the probe(s) aimed atdetecting RNA molecule(s).

Detected RNA molecule(s) can be polycistronic RNA, RNA corresponding totranscribed fragments of genomic DNA (nuclear and/or mitochondrialgenomic DNA), processed or unprocessed RNA(s) in said cell.

In a particular embodiment of the invention, the first probe aimed atrevealing a gDNA target region is a single stranded DNA fragment rangingin size from 80 bp to 1000, 2000 or 3000 bp. Such a probe can range insize from 90 to 150, 200, 300 or 500 bp, in particular from 95 to 110,120 or 130 bp, preferably sizing 99 bp. According to a particularembodiment, the first probe ranges in size from 200, 400 or 600 bp to900, 1000, 1100 or 1300 bp. In a specific embodiment, the firstnucleotide probe is a single stranded DNA fragment ranging in size from80 to 3000 bp, or from 80 to 2000 bp or from 80 to 1200 or to 1500 bp.

FIG. 22 discloses experiments demonstrating that the efficiency of the3D-FISH/mTRIP method of the invention is not affected by the size of theprobe.

Therefore, the invention also relates to nucleic acid molecules for useas probes, in particular as first probes according to the presentinvention, which are specific for the segment of mitochondrial gDNAlocalized between nucleotide position 446 and nucleotide position 162024on the H strand of the mt genome (SEQ ID NO:19) or are specific for asegment having 80% identity with such a segment (SEQ ID NO:19), or arespecific for fragments thereof. According to a particular embodiment,said nucleic acid molecules are specific for the segment ofmitochondrial gDNA between nucleotide position 446 and nucleotideposition 16366 (numbered with respect to the H strand of the mt genome)of the mitochondrial genome of a human eukaryotic cell. According toanother embodiment, nucleic acid molecules are complementary to anucleic acid sequence that has at least 80% identity with a target DNAregion localized between nucleotide position 446 and nucleotide position162024 on the H strand of the mt genome, in particular nucleotideposition 446 and nucleotide position 16366, of the mitochondrial genomeof a human eukaryotic cell.

Nucleic acid molecule of the invention may therefore be selected amongthe groups of:

-   -   i. a nucleic acid molecule comprising SEQ ID NO: 17 and SEQ ID        NO:

18, in particular a nucleic acid molecule complementary to or at leastcomplementary to or which hybridizes with the sequence of mt DNA or fromnucleotide position 425 to nucleotide position 16366 on the H strand ofthe mt genome, or

-   -   ii. a nucleic acid molecule whose sequence is framed by SEQ ID        NO: 1 and SEQ ID NO: 17, in particular as disclosed in SEQ ID        NO:19, and which is complementary to or at least partly        complementary to or hybridizes with the sequence of mt gDNA from        nucleotide position 446 to nucleotide position 225 on the H        strand of the mt genome, said nucleic acid molecule having from        80 to about 400 nucleotides; or    -   iii. a nucleic acid molecule whose sequence is framed by SEQ ID        NO: 1 and SEQ ID NO: 18, in particular as disclosed in SEQ ID        NO:19, and which is complementary to or at least partly        complementary to or hybridizes with the sequence of mt gDNA from        nucleotide position 446 to nucleotide position 16366 on the H        strand of the mt genome, said nucleic acid molecule having from        80 to about 800 nucleotides; or    -   iv. a nucleic acid molecule whose sequence comprises, in this        order, SEQ ID NO: 1, SEQ ID NO:17 and SEQ ID NO: 18, in        particular as disclosed in SEQ ID NO:19, and which is        complementary to or at least partly complementary to or        hybridizes with the sequence of mt gDNA from nucleotide position        446 to nucleotide position 16024 on the H strand of the mt        genome, said nucleic acid molecule having from 80 to about 1200        nucleotides.

According to a particular embodiment, a probe according to the presentinvention is specific for the segment of mitochondrial gDNA localizedbetween nucleotide position 544 and nucleotide position 162024 on the Hstrand of the mt genome (SEQ ID NO:1 concatenated with SEQ ID NO:19) oris specific for a segment having 80% identity with such a segment (SEQID NO:1 concatenated with SEQ ID NO:19), or is specific for fragmentsthereof.

According to another embodiment, a nucleic acid molecule probe of theinvention is complementary to a nucleic acid sequence that has at least80% identity with a target DNA region localized between nucleotideposition 544 and nucleotide position 162024 on the H strand of the mtgenome, in particular nucleotide position 544 and nucleotide position16366, of the mitochondrial genome of a human eukaryotic cell.

Considering the second probe aimed at revealing RNA molecule(s),according to a particular embodiment of the invention, said second probeis a single stranded nucleotidic DNA fragment ranging in size from 100bp to 3000 bp, and preferably sizing between 100, 200, 300 bp and 1000,1200, 1500, 2000 bp when aimed at detecting mitochondrial transcripts.

According to a particular embodiment, the size and/or sequence of secondprobe(s) aimed at revealing RNA molecule(s) is particularly adapted toenable the detection of transcription products resulting from thetranscription of coding segments of genomic DNA, especially segmentscorresponding to genes.

As stated above, in a particular embodiment, the nucleic acid moleculesuitable for use as first nucleotide probe is specific for a segment ofa non transcribed mitochondrial gDNA, especially an entirelynon-transcribed mitochondrial gDNA segment, according to the definitionprovided herein.

Accordingly, the first nucleotide probe or molecule is complementary ofa genomic DNA region that has no corresponding RNA transcript at acellular level, and therefore remains RNA-free at the cellular level.

In a particular embodiment, the first nucleotide probe or moleculetargets the genomic DNA sequence localized between the two promoters PH1(or HSP—Heavy Strand Promoter) and LSP (Light Strand Promoter) of themitochondrial genome of a eukaryotic cell. Both the HSP and LSPpromoters are found in all eukaryotic mtDNA although their name mightdiffer depending on the species to which the considered eukaryotic mtDNAbelongs. The corresponding names can be identified through theliterature.

The respective position (coordinates) of these two promoters on thehuman mitochondrial genome (NCBI, GenBank or MITOMAP sequence referenceNC_012920.1) is indicated in Table 2.

In the human mitochondrial genome, such a first probe or nucleic acidmolecule sequence has been designated mREP by the inventors. Coordinatesof this sequence are given in Table 1 (NCBI, GenBank or MITOMAP sequenceNC_012920.1, used as reference). mREP (SEQ ID No 1) is also disclosed inFIG. 13 a.

In another embodiment, said first probe comprises the mREP sequence (SEQID No 1).

In a specific embodiment, the first nucleotide probe or molecule iscomplementary to a nucleic acid sequence that has at least 80% identitywith the target DNA region that is localized between the two promotersPH1 (or HSP) and LSP of the mitochondrial genome of a eukaryotic cell,or has at least 80% identity with mREP.

By “at least 80% identity” it is meant that their sequence ofnucleotides differ from less than 20%, calculated over the entire lengthof the considered sequence (global alignment calculated for example bythe Needleman and Wunsch algorithm). The complementarity is similarlydetermined by such a global alignment. In a particular embodiment, forexample when compared sequences substantially differ in their length,identity and complementary can be determined using the same cutoffvalues by using a local alignment calculated for example by the Smithand Waterman algorithm. The modifications of nucleotides are especiallysubstitutions.

A sequence which is said to be partly complementary to a sequence ofreference may be a sequence with “at least 80% identity” with saidsequence of reference.

The genomic DNA sequence localized between the two promoters PH1 (orHSP) and LSP of the mitochondrial genome of a eukaryotic cell is part ofa highly variable region in the mitochondrial genome. FIG. 13 bdiscloses polymorphism variations known to date with respect to thehuman mREP sequence (SEQ ID No 1) of the invention. However, very few ofthese variations can be found simultaneously in an individual, i.e. only1, 2, 3 or 4 of these variations can be found simultaneously. Saidvariations are generally linked to a subpopulation type (e.g. Caucasian,African . . . ). These polymorphism variations are a basis for thedesign of variants of mREP.

Table 3 discloses the percentages of identity between the sequencescorresponding to the mREP probe (SEQ ID No 1) in several organisms andspecies, with respect to human mREP probe, along with coordinates ofsaid sequences on the corresponding mitochondrial genomes (SEQ ID No 2to 16).

TABLE 3 Percentages of identity between the sequences (SEQ ID No 2 to16) corresponding to the mREP (SEQ ID No 1) probe in several organismsand species. Refseq are NCBI or GenBank reference numbers. AccessionmtDNA number Genome mREP alignment Organism Refseq size (bp) coordinatesPrimates Homo sapiens NC_012920.1 16 569 446-544 Pan troglodytesNC_001643.1 16 554 16424-16521 Pan paniscus NC_001644.1 16 56316433-16530 Gorilla gorilla NC_001645.1 16 364 16233-16332 Pongopygmaeus NC_001646.1 16 389 16247-16357 Hybolates lar NC_002082.1 16 47216148-16246 Cebus albifrons NC_002763.1 16 554 15946-16044 Othermammalians Capra hircus NC_005044.2 16 643 15519-15630 Mus musculusNC_005089.1 16 299 15654-15757 Oryctolagus cuniculus NC_001913.1 17 24515767-15867 Canis lupus NC_008092.1 16 729 16456-16555 Rattus sordidusNC_014871.1 16 309 15643-15744 Felis catus NC_001700.1 17 009 759-864Castor canadensis NC_015108.1 16 701 15766-15866 Avian Gallus gallusNC_001323.1 16 775 473-571 Fish Danio rerio NC_002333.2 16 596 682-782

In a specific embodiment, the first nucleotide probe comprises a nucleicacid molecule having the sequence of any one of SEQ ID No 1, SEQ ID No2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQID No 8, SEQ ID No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12, SEQ IDNo 13, SEQ ID No 14, SEQ ID No 15, SEQ ID No 16, or the sequence that iscomplementary of any one of SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12, SEQ ID No 13, SEQ ID No 14,SEQ ID No 15, SEQ ID No 16, or is a fragment of said sequences.

In a specific embodiment, the first nucleotide probe comprises a nucleicacid molecule encompassing fragments of the nucleic acid sequence of anyone of SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5,SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10, SEQ IDNo 11, SEQ ID No 12, SEQ ID No 13, SEQ ID No 14, SEQ ID No 15, SEQ ID No16, or the nucleic acid sequence that is complementary of any one of SEQID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ ID No6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10, SEQ ID No 11,SEQ ID No 12, SEQ ID No 13, SEQ ID No 14, SEQ ID No 15, SEQ ID No 16.

In a specific embodiment, the first nucleotide probe consists of anucleic acid molecule that has the nucleic acid sequence of any one ofSEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ IDNo 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10, SEQ ID No 11,SEQ ID No 12, SEQ ID No 13, SEQ ID No 14, SEQ ID No 15, SEQ ID No 16, orthe nucleic acid sequence that is complementary of any one of SEQ ID No1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQID No 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No12, SEQ ID No 13, SEQ ID No 14, SEQ ID No 15, SEQ ID No 16.

According to a particular embodiment, the first nucleotide probe is anucleic acid that has at least 80% identity with the nucleic acidsequence of any one of SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, SEQID No 10, SEQ ID No 11, SEQ ID No 12, SEQ ID No 13, SEQ ID No 14, SEQ IDNo 15, SEQ ID No 16, or the nucleic acid sequence that is complementaryof any one of SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ IDNo 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10,SEQ ID No 11, SEQ ID No 12, SEQ ID No 13, SEQ ID No 14, SEQ ID No 15,SEQ ID No 16.

In a particular embodiment, the first nucleotide probe encompasses a mixof more than one, but distinct probes, especially probes comprisingnucleic acid molecules having distinct sequences from each other, chosenamong the sequences disclosed above. In a specific embodiment, the firstnucleotide probe encompasses a mix of two nucleic acid molecules havingsequences that are complementary to each other. Such a mix might have tobe denaturated prior to its use.

FIG. 13 b discloses polymorphism variations known to date with respectto the mREP sequence. These polymorphism variations may be used todesign probes alternative to mREP corresponding to SEQ ID No 1.

FIG. 13 c discloses alignments between the human mREP sequence and thecorresponding sequences in different organisms, which are also disclosedherein under SED ID No 2 to 16.

According to a particular embodiment of the invention, when bothmitochondrial gDNA and RNA molecules are detected, the at least secondnucleotide probe detecting RNA molecule(s) targets RNA/DNA hybridmolecule(s) or targets RNA hybridizing to the mitochondrial gDNA in theD-loop region of the mitochondrial gDNA.

In a specific embodiment, the second nucleotide probe is specific for aRNA molecule involved the formation of a RNA/DNA hybrid structure or afragment thereof, or is specific for a RNA hybridizing the gDNA in theD-loop region or a fragment thereof, and may comprises or consists ofthe sequence disclosed under SEQ ID NO 17 or SEQ ID NO 18, or be afragment thereof. In a particular embodiment the second nucleotide probehas at least 80% identity with SEQ ID NO 17 or SEQ ID NO 18. In aparticular embodiment the second nucleotide probe has the sequencedisclosed under SEQ ID NO 17 or SEQ ID NO 18.

SEQ ID NO:17 is the sequence of the PL-OH probe (position 225-425)according to the reference human mitochondrial sequence NC_012920.1,GenBank:

225 gtagga cataataata acaattgaat gtctgcacagccactttcca cacagacatc ataacaaaaa atttccaccaaaccccccct cccccgcttc tggccacagc acttaaacacatctctgcca aaccccaaaa acaaagaacc ctaacaccagcctaaccaga tttcaaattt tatcttttgg cggtatgcac tttta 425

SEQ ID NO:18 is the sequence of the 7S probe (position 16366-16566)according to the reference human mitochondrial sequence NC_012920.1,GenBank:

16366 catgg atgacccccc tcagataggg gtcccttgaccaccatcctc cgtgaaatca atatcccgca caagagtgctactctcctcg ctccgggccc ataacacttg ggggtagctaaagtgaactg tatccgacat ctggttccta cttcagggtcataaagccta aatagcccac acgttcccct taaataagac atcacga 16566

In a particular embodiment, the first nucleotide or the secondnucleotide probe is directly labelled, in particular with a fluorescentgroup.

Labelling can be achieved with groups or labels such as Biotin, digoxinand digoxigenin (DIG), alkaline phosphatase and fluorescent groups orlabels such as fluorescein (FITC), Texas Red and rhodamine orderivatives thereof, or dyes including coumarins, rhodamines,carbopyronins, oxazines or derivatives thereof or quantum dots,especially fluorescent quantum dots, or derivatives thereof.

By “directly labelled”, it is meant that the detection of the label doesnot require the intervention of another, i.e. secondary, chemical agentor compound, including an antibody, to be achieved. Such a directlabelling might improve the specificity of the labelling.

Labelling can be achieved through a commercial kit, for exampleaccording to a nick translation procedure. An example of such acommercial kit is the Nick Translation Atto NT Labeling kit fromJenaBioscience; comprising the following dyes: Atto425=blue,Atto488=green, Atto550=red.

In a particular embodiment, the first nucleotide probe comprisesmodified nucleotides, as disclosed herein.

The invention also relates to a method for the in situ hybridization anddetection of nucleotidic material within at least one eukaryotic cell,which comprises the steps of:

-   -   a. Fixation of said cell in 1 to 4% paraformaldehyde (PFA),        preferably 2% PFA for about 20 to 30 minutes, especially 30        minutes,    -   b. Permeabilization of said fixed cell with 0.5% to 1% Triton        X100 in PBS (Phosphate Buffered Saline Buffer) 1×, for about 5        to 10 minutes at 4° C., especially 5 minutes,    -   c. Denaturation of the nucleic acid contents of said        permeabilized fixed cell by heating at a temperature in a range        of 72 to 78° C., preferably 75° C., for 2 to 8 minutes,        preferably 4 to 5 minutes, especially 5 minutes,    -   d. Contacting the nucleic acid(s) in the cell treated according        to step (c) with nucleotide probe(s) defined herein as first        probe(s) and optionally second probe(s) to enable hybridization        of said nucleic acid(s) with said probe(s), wherein the probe(s)        has(have) a size ranging from 80 to 3000 nucleotides, or from 90        to 1000 nucleotides, in particular from 95 to 110 nucleotides,        said nucleotide probe(s) being contained in an hybridization        solution comprising from 100 ng/μl to 10 μg/μl of salmon sperm        DNA,    -   e. Detecting the nucleic acid(s) hybridized to the probe(s)        added in step (d).

The denaturation step of said method can further be carried out in anappropriate buffer and/or with a chemical agent aimed at partiallydenaturing nucleic acids, as disclosed herein.

According to a specific embodiment of the invention, the denaturationstep of the nucleic acid contents of a fixed cell is carried out byheating at a temperature in a range of 72 to 78° C., preferably 75° C.,for 2 to 8 minutes, preferably 4 to 5 minutes, especially 5 minutes, inthe presence of a chemical agent such as formamide, especially asolution of 70% formamide or 70% formamide/2×SSC.

Said method can further comprise a step of washing the cell(s) contactedwith the nucleotide probe(s) with an appropriate buffer prior to thedetection step.

According to a particular embodiment, the hybridization step is carriedout during about 15 hours at 37° C.

According to a particular embodiment, the method of the inventionfurther comprises steps enabling labelling and detection of at least oneprotein of interest within the eukaryotic cell, in particular byimmunofluorescence.

The detection can especially be carried out by single-cell imaging.

To this end, the cell(s) or tissue(s) under assay can be contacted withantibodies specific for the protein(s) of which detection is sought.

According to a particular embodiment, the detection of nucleotidicmaterial and of the protein of interest is achieved in one step, inparticular simultaneously.

A step of analysis of the result(s) of the detection(s) might besubsequently performed.

Fixation of cells can alternatively be performed with agents such asparaffin, acetone, methanol, ethanol, a combination of methanol andacetone a combination of methanol and ethanol, formalin, a combinationof paraformaldehyde and methanol, or any combination(s) of the agentsdisclosed herein.

Said steps that can be involved in a method according to the inventionare detailed hereafter.

1. Cell(s) or Tissue(s) Fixation

According to a particular embodiment, the fixation of cell(s) ortissue(s) is performed on glass slide(s) with a solution of 1 to 4%paraformaldehyde (PFA), preferably 2% PFA for about 20 to 30 minutes,especially 30 minutes. According to a specific embodiment, the fixationis carried out with a solution of 2% PFA for about 30 minutes. Saidfixation can be carried out at room temperature, RT.

After the fixation, storage of the fixed material can be performed in abuffer such as PBS 1× (during maximum one year at 4° C.).

Alternatively, fixation can be carried out according to any one of thefollowing protocols:

-   -   Acetone Fixation (Fix cells in −20° C. acetone for 5-10        minutes);    -   Methanol Fixation (Fix cells in −20° C. methanol for 5-10        minutes);    -   Ethanol Fixation (Fix cells in cooled 95% ethanol, 5% glacial        acetic acid for 5-10 minutes);    -   Methanol-Acetone Fixation (Fix in cooled methanol, 10 minutes at        −20° C.; Remove excess methanol);    -   Methanol-Acetone Mix Fixation (1:1 methanol and acetone        mixture.; Make the mixture fresh and fix cells at −20 C for 5-10        minutes);    -   Methanol-Ethanol Mix Fixation (1:1 methanol and ethanol mixture,        Make the mixture fresh and fix cells at −20 C for 5-10 minutes);    -   Formalin Fixation (Fix cells in 10% neutral buffered formalin        for 5-10 minutes);    -   Paraformaldehyde-Methanol Fixation (Fix in 4% paraformaldehyde        for 10-20 minutes, Rinse briefly with PBS, Permeabilize with        cooled methanol for 5-10 minutes at −20° C.

While cell(s) or tissue(s) fixation is generally not performed at thisstage in standard DNA FISH procedures, standard RNA FISH proceduresrequire the fixation of cell(s) or tissue(s) with 4% PFA for 10 minutesminimum.

2. Cell(s) or Tissue(s) Permeabilization

In particular embodiments, the permeabilization step might be requiredto allow a good infiltration of the probes, especially through cellmembranes, in order for the probes to reach their target sequence. Knownchemical reagents used for permeabilizing cells in the prior art areHCl, detergents such as Triton or SDS or Proteinase K.

According to a particular embodiment of the invention, fixed cell(s) ortissue(s) are permeabilized using a 0.5% to 1% Triton X100 solution inan appropriate buffer, such as PBS 1×, for example during 5 min. Saidpermeabilization can be carried out at 4° C.

According to a particular embodiment, fixed cell(s) or tissue(s) can bewashed up to 3 times in PBS 1× prior to the permeabilization, and up to4 times in PBS 1× after the permeabilization.

According to a particular embodiment, the permeabilization can befollowed by incubation of the cell(s) or tissue(s) in 50%Formamide/2×SSC (saline-sodium citrate buffer) in PBS 1× at RT during 30min, and further switching to 70% Formamide/2×SSC just before thedenaturation.

Optionally, a control assay for the probe specificity can be performedby incubating the samples during 1 hour at 37° C. in RNase or DNasesolution (100 ug/ml), with an additional washing up to 3 times in PBS 1×prior to the incubation of the cell(s) or tissue(s) in Formamide.

Standard DNA FISH procedures generally involve a heat treatment of theglass slides (for example during 90° C. 1 h30 or at 37° C. overnight) toremove all enzymes that could interfere with the experiment, and thepermeabilization is achieved by incubation with 0.005% Pepsin/0.001M HClat 37° C. for 15 min, washing in (4%) paraformaldehyde/PBS, thenincubating in ethanol series: 70%, 90%, 100%, further followed by RNasetreatment that is performed to remove primary transcripts and subsequentwashing and dehydratation in ethanol series. The samples are finally airdried.

Standard RNA FISH procedures generally require permeabilization withTriton X100 0.5% in buffer (the composition of which might containPIPES, MgCl2, sucrose or NaCl . . . ), washing in 4% PFA/PBS for 10 minon ice and further washings (twice) in 70% ethanol and subsequentdehydratation in ethanol series: 80%, 95%, 100% ethanol and air dryingof the samples on a heating plate at 42° C.

To the contrary, the method of the invention in conducted in absence ofenzymatic (i.e. pepsin), acidic (i.e. HCl) or alkaline treatment,alcoholic treatment (i.e. ethanol) or drying agent or treatment (i.e.air dry).

3. Denaturation

The denaturation step is performed by heating the samples comprising theassayed cell(s) or tissue(s) at a temperature in a range of 72 to 78°C., preferably 75° C., for 2 to 8 minutes, preferably during 4 to 5minutes, especially 5 minutes. According to a specific embodiment, thesamples are heated at 75° C. during 5 minutes. The samples can then bekept on ice until the probe(s) are ready.

While the denaturation step is generally not performed in standard RNAFISH procedures, because such a step would be unnecessary since RNAs aresingle stranded molecules, standard DNA FISH procedures generallyinvolve heating the samples at temperatures close to 80° C., and theassistance of other treatments such as chemical or physical treatmentsusually used to denaturate nucleic acids (e.g. washing with 70% ethanolon ice or dehydratation with ethanol series) in order to obtain singlestranded DNA molecules.

However, the denaturation step according to the invention may be carriedout in the presence of chemical agent(s) aimed at partially denaturingnucleic acids, or in an appropriate buffer, such as formamide.

4. Probe Design and Preparation

According to the invention, probe(s) are preferably small in size (3 kbor shorter, preferably 1 kb or shorter) and the direct use offluorescence probe(s) for labelling DNA(s) and RNA(s) is preferred.

When a step of nick translation (direct fluorescence) andBiotin/Digoxigenin (undirect fluorescence) is necessary for probelabelling, thus further requiring primary and secondary fluorescentantibodies to reveal Biotin/Digoxigenin-DNA or RNA probe hybridization,a kit such as the Jena Nick Translation kit can be used to label theprobe(s) (Atto fluorescence) and the quantity of DNA recovered afterpurification should be estimated (about 10% loss compared to input).

5. Pre-Hybridisation

According to a particular embodiment, a pre-hybridization step can beperformed, comprising the following steps:

For 1 slide, mix in 25 μL final volume:

-   -   40 ng fluorescent probe (final concentration: 1.6 ng/μl)    -   400 ng salmon sperm DNA (from 100 ng/μl solution)    -   Buffer: 10% dextran sulfate/50% Formamide/2×SSC in PBS 1×

Incubate the mix 10 min at 80° C. in the dark

Optionally, pre-hybridized slides can be put to pre cool 30 min at 37°C. in the dark before incubating slides.

Standard DNA and RNA FISH procedures generally require a higherconcentration of fluorescent or tagged probe (final concentration ofabout 10 ng/μl) and the addition of Cot-1 DNA and Salmon Sperm DNA at afinal concentration of more than 0.5 μg/μL. The mix is then incubated (5to 10 min) at 74-85° C. in the dark.

According to a particular embodiment, the pre-hybridisation and/orhybridisation step is carried out in absence of Cot-1 DNA.

6. Hybridization

According to a particular embodiment of the invention, the hybridizationstep is be carried out during about 15 hours at 37° C. in a standardbuffer such as illustrated in the examples, e.g. an hybridization bufferwith 50% formamide, 10% dextran sulphate, in 2×SSC pH 7.0 or anothersimilar buffer as appropriate.

More specifically, the hybridization step can be done by incubating thesamples about 15 hours at 37° C. on a heating metal block in the dark.Standard DNA and RNA FISH procedures generally require overnighthybridization.

7. Washes and Mounting

According to a particular embodiment, the method of the inventionfurther comprises a step of washing the cell contacted with thenucleotide probe(s) with an appropriate buffer prior to the detectionstep.

For example, washing can be performed up to 2 times for 2 min in 3 mL2×SSC at RT (cover with black top to protect from light), then up to 2times in 1×SSC at RT, then up to 2 times in 0.1×SSC at RT. Finally, anultimate washing can be performed up to 2 times in PBS 1× at RT (all inthe dark).

At this point a simple 2D imaging can be performed. In order to obtain agreater resolution, 3D analysis can be preferred.

8. 3D-FISH to Achieve mTRIP (Mitochondrial Transcription and ReplicationImaging Protocol)

When 3D-FISH is performed, the following steps may be carried out.

Incubate 1 h in Hoechst 33342 10 ug/mL final

Wash 5 times in PBS 1×

Mount on 20 uL PBS 1× on 70% Ethanol cleaned and dried slides.

Keep the slides in Dark clean box at RT until confocal analysis.

As described until this point, the procedure allows the labelling ofgenomic DNA, especially mtDNA and RNAs. Further steps are required foradditional labelling of mitochondrial or cellular proteins.

According to an advantageous embodiment, the method of the inventionfurther comprises steps enabling labelling and detection of at least oneprotein of interest within the eukaryotic cell treated for in situhybridization and detection of nucleotidic material. For this purpose,immunofluorescence can be used and the cell can be contacted withantibodies specific for the protein(s) of which detection is sought.Antibodies raised against a particular protein or epitope can beobtained according to usual techniques commonly used in theimmunological field.

In a particular embodiment, the mTRIP/3D-FISH method of the inventionenables to achieve the detection of the nucleotidic material and theprotein of interest in one step, in particular simultaneously.

9. 3D-FISH/mTRIP Coupled Immunofluorescence:

The following steps might be performed:

Incubate the slide with BSA 5% in PBS 1× 1 h RT in the dark.

Wash 2 times in PBS 1×

Incubate with Primary antibody in BSA 1%, PBS 1×, 1 h RT dark

Wash 3 times in PBS 1×

Incubate with Secondary antibody in Hoechst 33342 10 μg/ml final,

BSA 1%, PBS 1×, 1 h RT dark

Wash 5 times in PBS 1×

Mount on 20 μL PBS 1× on 70% EtOH cleaned and dried slides.

Keep the slides in dark clean box at RT until confocal analysis.

Example of a Protocol According to the Invention Detailing ParticularSteps Performed in a Particular Embodiment of the Method of theInvention

The following procedure is suitable for the detection of the occurrenceof initiation of replication events in the mitochondrial genomic DNA ofhuman cell lines and human primary fibroblasts as well as in cells ofother eukaryote organisms.

The design of mtDNA probe(s) used for tracking the occurrence ofinitiation of replication events has to be adapted to the sequence ofthe corresponding mitochondrial genomes (see Table 3 that givescorrespondence of the mREP probe in other organisms).

1. Cell Fixation

Cells Fixation on glass slides: 2% paraformaldhehyde or PFA for 30 minat room temperature, RT.

Storage in PBS 1× (maximum one year at 4° C.)

2. Cell Permeabilization

Wash 3 times in PBS 1×

Permeabilize 5 min at 4° C. in 0.5% Triton X100 in PBS 1×

Wash 4 times in PBS 1×

Optional (control assay for the probe specificity)

-   -   Incubate 1 h at 37° C. in RNase or DNase solution (100 ug/ml)    -   Wash 3× in PBS 1×

Incubate the cells in 50% Formamide/2×SSC in PBS 1× at RT 30 min

Switch to 70% Formamide/2×SSC just before the denaturation

3. Denaturation

denaturate for 4 to 5 min, in particular 5 minutes, at 75° C.

Keep on ice until probe is ready

4. Probe Design and Preparation

size of probes˜1 kb or shorter

direct use of fluorescence probes is preferred

5. Pre-Hybridisation

For 1 slide, mix in 25 μL final volume:

-   -   40 ng fluorescent probe (final concentration: 1.6 ng/μl)    -   400 ng salmon sperm DNA (from 100 ng/μl solution)    -   Buffer: 10% dextran sulfate/50% Formamide/2×SSC in PBS 1×

Incubate the mix 10 min at 80° C. in the dark

Pre cool 30 min at 37° C. in the dark before incubating slides

6. Hybridization

Drop 25 uL of pre-hybridization mix on square parafilm

Invert slides on drops

Incubate 15 hours at 37° C. on a heating metal block in the dark (coverwith plastic top to set the dark position)

7. Washes and Mounting

wash 2 times for 2 min in 3 mL 2×SSC at RT

cover with black top to protect from light.

wash 2 times in 1×SSC at RT, then 2 times in 0.1×SSC at RT,

wash 2 times in PBS 1× at RT (all in the dark).

8. 3D-FISH/mTRIP

The following protocol is optimized for 3D Z-scanning of mammalian cellsusing a confocal spinning disk microscope. Z-stacks of 200 nm. 3Dreconstruction using the IMARIS (Bitplane software).

Incubate 1 h in Hoechst 33342 10 ug/mL final

Wash 5 times in PBS 1×

Mount on 20 uL PBS 1× on 70% EtOH cleaned and dried slides.

Keep the slides in Dark clean box at RT until confocal analysis.

Until this point the procedure allows the labelling of mt DNA and RNAs.With the next steps it allows the additional labelling of mitochondrialor cellular proteins.

9. 3D-FISH/mTRIP Coupled Immunofluorescence

The following protocol is optimized for 3D Z-scanning of mammalian cellsusing a confocal spinning disk microscope. Z-stacks of 200 nm. 3Dreconstruction using the IMARIS (Bitplane software).

Incubate the slide with BSA 5% in PBS 1× 1 h RT in the dark.

Wash 2 times in PBS 1×

Incubate with Primary antibody in BSA 1%, PBS 1×, 1 h RT dark

Wash 3 times in PBS 1×

Incubate with Secondary antibody in Hoechst 33342 10 ug/ml final,

-   -   BSA 1%, PBS 1×, 1 h RT dark

Wash 5 times in PBS 1×

Mount on 20 uL PBS 1× on 70% EtOH cleaned and dried slides.

Keep the slides in dark clean box at RT until confocal analysis.

The invention also relates to nucleic acid molecule(s) suitable for useas probe(s) as defined herein and especially designated as the “firstprobe” for use in the process of the invention.

The invention is in particular directed to a nucleic acid moleculesuitable for use as a probe suitable for in situ hybridization targetingthe genomic DNA.

Therefore, the invention also relates to a nucleic acid moleculesuitable for use as a probe, hybridizing with a target region in aeukaryotic genomic DNA, wherein said target region comprises a nucleicacid sequence which has no identified corresponding annealing RNA in themetabolically active cell containing said eukaryotic genomic DNA andtherefore remains RNA-free during transcription and replication of saidDNA genome. According to a particular embodiment, such a nucleic acidmolecule hybridizes with said RNA-free nucleic acid sequence, and hasthe same length as said RNA-free nucleic acid sequence or is longer.

Nucleic acid molecule(s) or probe(s) disclosed above and herein withrespect to a method according to the invention are themselves part ofthe object of the invention. They are prepared and/or used as either asingle stand molecule or a double strand molecule of complementarysequences.

In particular, such nucleic acid molecules are specific for a segment ofnon transcribed mitochondrial gDNA and comprises or consists of:

-   -   i. the nucleic acid having the sequence of any one of SEQ ID No        1 to SEQ ID No 16 or,    -   ii. the nucleic acid that has a sequence that is complementary        of any one of SEQ ID No 1 to SEQ ID No 16 or,    -   iii. a fragment of (i) or (ii) or,    -   iv. a nucleic acid that has at least 80% identity with the        nucleic acid sequence of any one of SEQ ID No 1 to SEQ ID No 16        or the nucleic acid sequence that is complementary of any one of        SEQ ID No 1 to SEQ ID No 16 or fragments thereof,        said nucleic acid molecule being either a single stand molecule        or a double strand molecule of complementary sequences.

The invention also relates to a nucleic acid molecule, binding to a RNAmolecule or to a RNA/DNA molecule, said RNA or RNA/DNA molecules beingmolecules hybridizing with a segment of mitochondrial gDNA localized inthe mitochondrial gDNA D-loop region, wherein said nucleic acid moleculecomprises or has the sequence or is a fragment or has at least 80%identity with sequence of SEQ ID No 17, or comprises or has the sequenceor is a fragment or has at least 80% identity with the sequence that iscomplementary of SEQ ID No 18.

The invention also relates to a kit for carrying out in situhybridization on fixed cells, comprising a so-called first probe,consisting of a nucleic acid molecule, of the invention, and comprisingoptionally a so-called second probe, consisting of a nucleic acidmolecule, of the invention, hybridizing with a RNA molecule or RNA/DNAhybrid molecule, as disclosed herein.

The invention also relates to a kit comprising a probe, consisting of anucleic acid molecule, suitable for in situ hybridization targeting thegenomic DNA as disclosed herein, and comprising optionally a so-calledsecond probe, consisting of a nucleic acid molecule, of the invention,hybridizing with a RNA molecule or RNA/DNA hybrid molecule, as disclosedherein.

According to a particular embodiment, said kits further comprise probes,consisting of nucleic acid molecule(s), and/or antibody(ies) foradditionally detecting protein(s).

Kits to label and detect in a same cell or tissue DNA and optionally RNAand/or proteins are useful for the detection of the occurrence ofinitiation events in the genomic DNA replication in eukaryote cell(s) ortissue(s).

Said kits can further comprise instructions for use in a process fordetecting the occurrence of initiation of replication events in genomicDNA in a eukaryotic cell, according to a method of the invention asdisclosed herein.

Said kits can further comprise reagents necessary for carrying out sucha process, as disclosed herein.

Said kits can further comprise material, e.g measurement material, datacarrier(s), recording support(s), to collect or analyze the datameasured by a process according to the invention.

The present invention is of particular interest for analyzing theprocessing of DNA, RNA or metabolites in cell(s) or tissue(s), and/oranalyzing the dynamics of said cell(s) or tissue(s), and/or detectingspecific diseases.

The invention is of particular interest for providing means useful forin vitro analysis, in vitro detection and optionally subsequentdiagnosis of mitochondrial disease(s) or neoplasic diseases(s) orcancer(s) or in vitro detection or monitoring of myopathies. Theinvention is also of particular interest for in vitro analyzing, invitro detecting and optionally subsequently diagnosing mitochondrialdisease(s) or neoplasic diseases(s) or cancer(s)

With respect to the interest of analyzing or detecting the occurrence ofinitiation of replication events in a context of mitochondrialdysfunction, it is knowledgeable to notice that the physiology andmetabolism of mitochondria impact not only in the production of cellularenergy (ATP) but also in cell growth, cell differentiation, cellsignaling and death (apoptosis). Thus, mitochondrial misfunction isassociated with a variety of diseases (cancers, myopathies,neuropathologies, infections), and with the ageing process.

Mitochondrial diseases encompass cardiomyopathy, neuropathy, Retinitispigmentosa, encephalomyopathy, hepatopathy, hypotonia, Renaltubulopathy, Leigh syndrome, Barth syndrome, optic atrophy and Ataxia,Leukodystrophy, Diabetes, Kearns-Sayre syndrome.

Mitochondrial dysfunctions can lead to or are involved in type 2diabetes, Parkinson, Alzheimer, Atherosclerotic heart disease, stroke orcancers.

The method of the invention, the probes described herein and kitsencompassing said probes or permitting to carry out the method of theinvention are useful in clinical diagnosis protocols by contributing tomeans necessary to identify and/or to class diseases associated withmitochondrial dysfunctions according to the default in mitochondrialmtDNA processing, e.g. transcription and replication, or in mtDNAcontent. This includes genetic diseases (mtDNA depletion diseases) andcancers. The same can be also used to identify mtDNA depletion inducedby clinical treatment (i.e., long term treatment with anti-HIVnucleoside analogues deplete mtDNA) or impaired or abolished initiationof mtDNA replication.

The method of the invention, the probes or nucleic acid moleculesdescribed herein and kits encompassing said probes or nucleic acidmolecules or permitting to carry out the method of the invention can beused in the analysis and detection of mitochondrial disease(s) ordisease(s) resulting from mitochondrial dysfunction(s) or impairment, ordisease(s) resulting in mitochondrial dysfunction(s) or impairment.

The method of the invention is applicable to all eukaryotic cells,including human, mouse, insect, yeast, fish or plant cell as a diagnosistool or a biotechnological tool for exploring the functions of saidcells.

The method of the invention, the probes or nucleic acid moleculesdescribed herein and kits encompassing said probes or nucleic acidmolecules or permitting to carry out the method of the invention canalso be used in the analysis and detection of neoplasic diseases(s) orcancer(s).

The invention provides means useful for the detection and diagnosis ofneoplasic or tumoral cell(s) or tissue(s), and especially to distinguishsaid cell(s) or tissue(s) among healthy cell(s) or tissue(s).

The present invention is also of particular interest for testing thecytotoxicity of organic and chemical compounds, especially drugs.

The invention also relates to a method for in vitro detecting alteredmitochondrial activity in cells, comprising the step of detecting thelevel of mitochondrial initation of DNA replication with a first probeas disclosed herein and detecting the level of mitochondrial transcriptswith a second probe as disclosed herein.

Other examples and features of the invention will be apparent whenreading the examples and the figures, which illustrate the experimentsconducted by the inventors, in complement to the features anddefinitions given in the present description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1K. A modified 3D-FISH/mTRIP method reveals a perinuclearmitochondrial subpopulation. Efficiency and characteristics of mTRIPlabeling is also disclosed (A) 3D reconstruction of a dividing HeLa cellshows perinuclear distribution of the mitochondrial subpopulationlabeled with the mt DNA probe mix mTOT (red). The entire mitochondrialnetwork is labeled with MitoTracker (green) and the nucleus with Hoechst33342 (blue). On the right, magnification shows MitoTracker labeling(bottom), mTOT (middle) and merge (top). Scale bar=10 um. Zoom scalebar=3 um. (B) 3D FISH-reconstructed HeLa cells labeled with the mTOTprobe (red), with or without nuclease treatment (specified on the top ofeach panel; the arrow indicates that the second nuclease was added after1 h incubation with the first nuclease). Scale bar=10 um. (C)Fluorescence intensity quantification of mTOT, with or without nucleasetreatment, indicated on the X-axis (“then” indicates that tof he secondnuclease was added after 1 h incubation with the first nuclease) n=30.T-test, compared to untreated cells, (*) p<0.05; (**) p<0.01; (***)p<0.001. (D-G) Epitope conservation during mTRIP treatment for relevantantibodies used in this study. (D) Anti-TOM22 immunostaining and (E)fluorescence intensity quantification of HeLa cells treated asindicated, or untreated, before immunolabelling. IF: immunofluorescence;steps 1+2: permeabilization+incubation with formamide 50%; step 3:denaturation in 70% formamide; mTRIP: complete procedure. A mildtreatment with proteinase K (5 min at 37° C.) was used as control ofprotein degradation; this reatment does not completely degrade proteins(not shown) and is thus compatible with fluorescence labelling.Fluorescence intensity quantification of (F) Polγ and (G) TFAMimmunostaining of HeLa cells pre-treated with the complete mTRIPprotocol, or untreated. (H) Scheme for 3D analysis of immunolabelledcells. Confocal acquisition, 3D-reconstruction and quantification (seeMethods). TOM22 immunolabelling was used to define the mitochondrialmass. The same procedure was also used for the labelling of mtDNA probesby mTRIP. (I) mTRIP labels mitochondrial RNAs and DNA: 3D-reconstructionof a HeLa cell shows the mitochondrial fraction labelled with the mtDNAprobe mix mTOT (red). The mitochondrial network is labelled with TOM22by immunofluorescence (green) and the nucleus with Hoechst (blue).Below, 2.5× magnification of mTOT (left), and merge (right). Scalebar=10 μm. (J) 3D-reconstructed cells labelled with mTOT in presence orabsence of proteinase K treatment. The other key FISH probes used inthis work, mTRANS and mREP (which labels only transcripts and DNA,respectively, as described below in the text) were also tested. (K)Fluorescence intensity quantification of mTOT, mTRANS, and mREP, with orwithout proteinase K treatment; for each condition n=30, threeindependent experiments. T-test, compared to untreated cells (***)p≤0.001.

FIGS. 2A to 2D. Spatio-temporal distribution of DNA processingmitochondrial subpopulations (A) 3D-FISH/mTRIP of HeLa cells with 14individual mtDNA probes (red), each covering a portion of the entiremitochondrial genome. Nuclei (blue) are labeled with Hoechst 33342.Scale bar=10 um. The probe number/name, and the mitochondrial gene(s)covered by the probe are indicated on the top and on the bottom of eachpanel, respectively. The central panel is a schematic representation ofthe mt genome (external circle) and of single genes within, at scale.tRNA genes are indicated with the corresponding letter. All genes arelocated on the H-strand, with the exception of ND6, located on theL-strand. The ribosomal RNAs (16S and 12S) are in dark grey and areslightly shifted out of the circle. The D-loop region that contains theorigin of replication of the H-strand (O_(H)) and the promoters of boththe H and the L strands (PH₁-PH₂, and PL, respectively) is shown inblack and shifted out of the circle. The inner circle represents theposition of each individual probe (see coordinates in Tables 1 and 2);some probes overlap by a few dozen of bases with the neighbor probes.The 14 individual probes cumulatively cover the complete mt genome (B)Fluorescence intensity quantification of 3D-reconstructed HeLa cellslabeled with each of the individual 14 mtDNA probes, indicated on theX-axis, untreated or treated with either DNase I and RNase A. Key genesand regulatory regions are indicated on top. n=30, from threeindependent experiments. For each probe, the t-test was performed fornuclease-treated versus untreated cells; (*) p<0.05; (**) p<0.01; (***)p<0.001. A further probe, called ND6, which covers the gene with thesame name located on the L-strand, was also tested. For most probes, thelabeling decreases dramatically or almost disappears after RNasetreatment, indicating that the labeling essentially target RNAmolecules. A partial or total reduction of the labeling results fromDNase treatment of probes 4, 8 and 13, indicating that these probestarget also mtDNA. Probes 4 and 13 cover regions of the mt genome thatcontains replication origins (O_(L) and OH, respectively), indicated onthe bottom. (C) 3D FISH/mTRIP-reconstructed IMR90 primary fibroblastslabeled with the 14 individual mtDNA probes (red); legend elements as in(a). (D) Fluorescence intensity quantification of the 14 individualmtDNA probes; legend elements as in (B). n=30 from three independentexperiments.

FIGS. 3A to 3E. Detection of regions containing mitochondrialreplication origins. (A) Schematic localization of DNA and RNA labelingby mt probes used in 3D-FISH assay. DNA labeling is observed on threeregions (not at scale), two of which correspond to the major origins ofreplication of the mt genome (O_(H) and O_(L), probes 13 and 8,respectively), and the third (probe 8) to a additional O_(L) origin ofreplication, that was previously identified¹³. The distances between mtorigins are indicated. (B) Characterization of the O_(H) region analysedby 3D-FISH with progressively shorter probes. The region covered by eachprobe is shown on the left panel that also indicates the main geneticelements present in the region (LSP=light-strand promoter; P_(H1) andP_(H2) stand for heavy-strand promoter 1 and 2, respectively;O_(H)=origin of H strand DNA replication; F=tRNA^(Phe); 12S=12S rRNA,P=tRNA^(Pro)). Only the probe mREP covers a DNA region that issubstantially not transcribed (indicated as RNA-free), while probes 13and 13-1 cover also transcribed regions. Panels on the right show by3D-FISH/mTRIP the localization of mitochondrial entities labeled witheach of the probes (red); nuclei in blue (Hoechst 33342). Scale bar=10um. (C) 3D FISH-reconstructed HeLa cells labeled with mREP (red) andmTRANS (green) probes, with or without nuclease treatments. Scale bar=10um (upper panels), and fluorescence intensity quantification (lowerpanels). Values of mREP in the presence of DNaseI and of mTRANS in thepresence of RNaseA correspond to background. n=30 from three independentexperiments: t-test for nuclease-treated versus untreated cells; (***)p<0.001. (D) 3D-FISH/mTRIP coupled IF with mREP (red) probe andanti-Poly (green). On the right, fluorescence intensity quantificationof mREP-positive and mREP-negative Poly labeled areas. Examples of therespective areas are shown on the bottom, circled, lower panels: Polγ;upper panels: merge. n=300 from three independent experiments; T-test(***) p<0.001. (E) 3D-FISH/mTRIP coupled IF with mREP (red) probe andanti-TFAM (green). On the right, fluorescence intensity quantificationof mREP-positive and mREP-negative TFAM-labeled areas. Examples of therespective areas are shown on the bottom, circled, lower panels: TFAM;upper panels: merge. n=300 from three independent experiments; T-test(***) p<0.001.

FIGS. 4A to 4C. 3D-FISH reveals mitochondria lacking transcription andinitiation of replication activity. (a) 3D-FISH/mTRIP reconstructed HeLacell also immunolabeled for the mitochondrial protein TOM22 revealssimultaneously mtDNA, mtRNA and the mitochondrial network. Right panel:mREP; middle panel: mTRANS; left panel: merge. In the left panel,mitochondrial transcripts (mTRANS, red, see arrow) are detected asindependent entities in the mitochondrial network (anti-TOM22, green),whereas the mt initiation of replication units (probe mREP, blue)essentially colocalize with the transcript carrying units (merge,purple, see arrowheads).

FIGS. 5A to 5B. Characterization of the 3D-FISH labeling (A)3D-FISH/mTRIP reconstructed HeLa cells labeled with: the mTOT probe(red) at saturating concentrations (200 ng; left panel); co-FISH of mTOTprobe (red) and of the Hs Alu probe, for the human nuclear Alu sequence,(green) (middle left panel); mTOTΔr, that consists in mTOT withoutprobes 1, 2, and 14 that cover the mt ribosomal genes, untreated (middleright panel), and treated with RNAse A (right panel). Scale bar=10 um.(B) Fluorescence intensity quantification of mTOT and mTOTΔr with andwithout nuclease treatment. n=30 from three independent experiments;T-test, compared to untreated cells, (*) p<0.05; (**) p<0.01; (***)p<0.001.

FIGS. 6A to 6C. Real-time quantitative PCR of individual mitochondrialgenes and comparison with FISH data. (A) Expression levels of theindividual mitochondrial genes in Hela cells. 16S and 12S were analyzedwith two independent sets of primers. Mean of 3 experiments±standarddeviation. (B) Relative gene expression of 16S and ND1 compared to 12S(12S was arbitrarily indicated as 1) in HeLa cells and in IMR-90 humanprimary fibroblasts. (C) Fluorescence intensity quantification of probeND1 (and of probe 3, data from FIG. 2 B, used as comparison) in presenceand absence of nucleases. For each probe the coordinates in themitochondrial genome are indicated below. n=30, three independentexperiments. T-test, compared to untreated cells (***) p≤0.001. On theright, 3D-reconstructed HeLa cell labelled with probe ND1 whichrecognizes a portion of the ND1 gene; scale bar=10 μm.

FIGS. 7A to 7C. Semi-quantitative analysis of the proportion ofmitochondria labeled with each of the 14 mt DNA probes. (A)3D-FISH/mTRIP coupled to immunofluorescence. Reconstructed HeLa cellslabeled with the mtDNA probe indicated (red) and anti-TOM22 (green). Thenumber in each panel indicates the probe used. (B) Percentage of3D-FISH/mTRIP labelled mitochondria in the total mitochondrialpopulation (for each probe, co-labelling of 3D-FISH/mTRIP andanti-TOM22). For each probe, n=30 cells; three independent experiments.(C) Intensity of fluorescence in TOM22-labelled mitochondria, calculatedfor each probe by multiplying the intensity of fluorescence (fi) by thepercentage (p) of FISH labeled mitochondria (=fi×p). Each valueindicates the relative amount of transcripts carried by theTOM22-labelled mitochondrial population.

FIGS. 8A and 8B. 3D-FISH on the 12S region using an alternative probe.(A) 3D-FISH/mTRIP of two HeLa cells with the probe 14-1. Scale bar=10um. (B) Quantification of the fluorescence intensity with the probe 14-1(data for probes 1 and 14 are from FIG. 2 b ) in HeLa cells, indicatingthat the labeling is dramatically lower for the 12S (probes 14 and 14-1)than for the 16S (probe 1) containing region. n=30 from threeindependent experiments.

FIG. 9 . Perinuclear distribution of mitochondria labeled with theindividual mtDNA probes. The histogram shows the perinuclearlocalization, defined as the region within 2 μm from the nuclear border,of mitochondria labeled with the probes indicated on the X-axis, in HeLaells and in IMR-90 primary fibroblasts (n=30; from three independentexperiments). Mean±SEM.

FIGS. 10A to 10F. Co-labeling with several mtDNA probes. (A) 3DFISH/mTRIP-reconstructed HeLa cells co-labeled with probes 2 (green) 3(red) and 4 (blue), upper panels; and merge with 5× magnification ofthree distinct regions (lower panels). The probe used is indicated withon top. Scale bar=10 μm. (B) percentage of co-localization of thevarious probes; n=30 cells from three independent experiments.Co-labeling shows that most mitochondrial entities labeled with probe 2(16S RNA) are also labeled with probe 3 (ND1), the following gene on theH-strand, but not with probe 4 that covers ND2, the further gene on theH-strand. Percentages of co-labeling are measured taking into accountthe total intensity of fluorescence specific to each probe. Therefore,percentages of co-labeling between probe pairs (as probes 2 vs 3compared to probes 3 vs 2) may be different. (C) Scheme of the positionof probes on the mitochondrial genome, as from FIG. 2 a . (D) 3DFISH/mTRIP-reconstructed HeLa cells co-labeled with probes 7 (red) and 9(green), upper panels; with probes 10 (red) and 9 (green) middle panels;with probes 12 (blue) and 14 (green) (lower panels). The probes used areindicated on each panel. Merge are on the right panels. Scale bar=10 um.(E) Percentage of co-localization of the various probes; for each pairof primers, n=30 cells, from three independent experiments. Note thehigh intensity of labeling with probe 7 at some mitochondrial entities,which may explain the reduced percentage of co-labeling with probe 9.Note also the strong and distinct labelling with probe 10 compared tothe more diffuse labeling with probe 9, as well as the different spatialdistribution of the two types of labelling. These differences mayexplain the limited co-localization between probes 9 and 10. (F) Summaryof co-localization results. On the X-axis is indicated the tested probe.On the Y-axis is reported the intensity of co-labelling with the probeindicated on the segment of the column. These Y-values represent thepercent of colabeling multiplied by the fluorescence intensity signal ofthe tested probe.

FIGS. 11A and 11B. Analysis of probes to label initiation of mt DNAreplication. (a) 3D FISH/mTRIP-reconstructed HeLa cells labeled withprobe 13-1 (red) either untreated or treated with nucleases, asindicated. For each condition, two different cells are shown (upper andthe lower panel, respectively). Scale bar=10 um. (b) Fluorescenceintensity quantification of 3D-reconstructed HeLa labeled with eitherprobe 13, probe 13-1, or probe mREP, indicated on top. The X-axisindicates whether cells were untreated or treated with either DNase Iand RNase A. For each probe and for each condition, n=30 from threeindependent experiments. T-test, compared to untreated cells, (**)p<0.01; (***) p<0.001. Values for mREP, shown here for directcomparison, are as in FIG. 3B. Treatment with DNaseI induces an increasein the labeling with probe 13-1, indicating that a portion of this probeis normally inhibited from binding because of the presence of aDNA-associated structure. However, this is not the case with the moreextended probe 13. The reason for such increase is not clear and thesequence involved has not been identified.

FIGS. 12A to 12D. mREP labels initiation of mtDNA replication. (A)Labeling with the mREP probe anticipates oxydative stress-dependentincrease of the mtDNA content. Kinetics of 3D-FISH coupledimmunofluorescence (IF) labeling of H2O2-treated HeLa cells; anti-TOM22(green, upper panels) and either mREP (red, middle panels) or mTRANS(red, lower panels) probes. Scale bar=10 um. (b) Fluorescence intensityquantifications. TOM22; mREP and mTRANS. n=30 from three independentexperiments. mtDNA content estimation by qPCR (12S region); expressionof the CytC transcript, coded by a nuclear gene, by qPCR. n=3. T-test(*) p<0.05; (**) p<0.01; (***) p<0.001. Ctrl, untreated. (C) mREP probecolocalizes to various extents with BrdU-positive mitochondria.3D-reconstructed cells labelled with mREP (red), anti-BrdU (green), andmerge; nuclei are labelled by Hoechst (blue). Scale bar=10 μm. Below,2.5× magnification of two regions, proximal and distal to the nuclearsurface are shown on right panels. Circles indicate representativemREP-positive and mREP-negative areas, where BrdU labelling was alsomeasured. (D) Fluorescence intensity measurement of BrdU labelling showshigher values in mREP-negative than in mREP-positive areas. n=300 areas,three independent experiments. (***) p≤0.001.

FIGS. 13A to 13C. mREP sequences and alignments. (a) Human mREP sequence(SEQ ID No 1), having coordinates 446-544 on the human mtDNA sequencedisclosed under accession number NC_012920.1 (NCBI or GenBank or MITOMAPaccession number) (b) mREP (SEQ ID No 1)—Human polymorphism—variations.Positions where polymorphisms, e.g. nucleotide(s) variation(s), might befound are put on a black background. One skilled in the art can identifyvariable positions in the mREP sequence from the above-mentionedindications (c) Human mREP alignments with the corresponding sequences(SEQ ID No 2 to 16) in different organisms (Accession numbers disclosedin said figure are NCBI accession numbershttp://www.ncbi.nlm.nih.gov)/).

FIGS. 14A to 14E. (A) Experiment disclosing a tight association of mtinitiation of DNA replication and mt transcripts in healthy primarycells but not in cancer-derived cell-lines: 3D-FISH/mTRIP-reconstructedHeLa cell (upper panel) and primary human fibroblast IMR-90 (lowerpanel) also immunolabeled for the mitochondrial protein TOM22.Mitochondrial transcripts (probe mTRANS, red) are detected asindependent entities in the mitochondrial network (anti-TOM22, green),whereas the mt initiation of replication units (probe mREP, blue)essentially colocalize with the transcript carrying units (merge,purple). Right panels represent details of the left panels (5×magnification). White arrows indicate purple foci where mTRANS and mREPco-localize (col-localization of transcription and initiation ofreplication signals). Note that in primary fibroblasts mitochondrialtranscripts essentially co-localize with initiation of replication unitsand are almost not detected as independent units. Scale bar=10 μm. (B)Quantification of co-labelling of either mTRANS or mREP with anti-TOM22in HeLa cells and IMR-90 primary fibroblasts expressed in percentage ofcol-labelling. (C) Quantification of co-labelling of mTRANS with mREP(mitochondrial entities carrying transcripts and being involved ininitiation of replication) white columns, and of mREP with mTRANS(mitochondrial entities involved in initiation of replication that alsocarry transcripts), grey columns in Hela cells (left panel) and IMR-90primary fibroblasts (right panel). For each condition, n=30 cells; threeindependent experiments. (D) Expression level of mitochondrialtranscripts in cancer cells (E) Expression level of mitochondrialtranscripts in healthy cells. For each condition, n=30 cells; threeindependent experiments.

FIGS. 15A to 15F. Analysis of mtDNA regulatory regions by mTRIP (A)Fluorescence intensity quantification of probes PL-OH (left) and 7S(right) in presence or absence of indicated nucleases; n=30, threeindependent experiments: t-test for nuclease-treated versus untreatedcells; (***) 0.001. (B) Schematic representation (not to scale) ofD-loop region of mtDNA analysed by mTRIP, key elements and coordinatesin mtDNA are indicated; vertical grey bars in O_(H) region represent thethree CSB sites; 16S and 12S genes, and 7S region are shown. FISH probes(horizontal black bars) are named and their position on mtDNA isindicated. Below are summarized detected nucleic acids in thecorresponding region on scheme, including data of probe mREP from FIG.3D and of probe PH1-2 from panel E (++: exclusive detection; +:detection, −: no detection; nd=not done). (C) 3D-reconstruction of cellslabelled with PL-OH, 7S, and merge. Cells were treated with thenucleases indicated above or untreated. In merge panels arrowheads andarrows indicate foci with the prevalent (1) and alternative (2-6)labelling patterns, respectively, according to the sensitivity to thevarious nucleases. (D) Nucleic acids detected at large PL-OH and 7Sfoci, with the pattern number indicated as in panel C (a/o=and/or). (E)Upper panels: 3D-reconstruction of cells labelled with probes PH1-2 inthe presence and in the absence of the indicated nuclease. On the right,fluorescence intensity quantification; n=30, three independentexperiments: t-test for nuclease-treated versus untreated cells; (***)p≤0.001. Lower panels: 3D-reconstruction of cells colabelled with PH1-2,probe 1, and merge. Percentage of colocalization between probes is shownon the right. (F) Graphic summarizes intensity of fluorescence labellingwith different probes in presence or absence of nucleases, normalized tovalues of mREP (in absence of nucleases).

FIGS. 16A and 16B. mTRIP detects mtDNA processing alterations in cellswith perturbed mtDNA content. (A) Upper panels: 3D-reconstructed HeLacells analysed by 3D-FISH/mTRIP with probes mREP and mTRANS and byimmunofluorescence with TOM22 (colour indicated on each panel). Cellswere either untreated or treated with EtBr for 3 days to decrease theirmtDNA content. Note significant variation in distribution and aspect ofmREP and mTRANS labelling in these cells compared to untreated cells.HeLa rho⁰ cells were also analysed. Scale bar=10 μm. Fluorescenceintensity quantification is shown in lower panels; n=30, threeindependent experiments. mtDNA content of cells analysed by qPCR in the12S region, and expression levels of 16S and CytB RNAs analysed byRT-qPCR are shown. T-test for treated or mutated cells versus untreatedcells, (***) p≤0.001. (B) 3D-reconstructed normal human primaryfibroblasts (IMR-90) and fibroblasts from a patient with mutated Rrm2banalysed by mTRIP with probes mREP and mTRANS, and by immunofluorescencewith TOM22. Results similar to IMR-90 were obtained with two other typesof human primary fibroblasts (not shown). Scale bar=00 μm. Fluorescenceintensity quantifications are shown on right; n=30, three independentexperiments. Quantification of the mtDNA content (12S region) by qPCR isshown below.

FIGS. 17A and 17B. Assays on patients with mitochondrial diseases. (A)mTRANS and mREP labelling on primary fibroblasts from patients withmitochondrial diseases (mtDNA depletion syndromes). Nuclei (blue) arelabelled by Hoechst (only with probe mREP) (B) Fluorescence intensityquantification of mTRANS ans mREP labelling in fibroblasts from a fewnormal individual (control) and from patients with mitochondrialdiseases (mtDNA depletion). The red line indicates the value ofcorresponding to the average controls. Note that in spite all thesepatients display severe mtDNA depletion, various levels of mTRANS andmREP are detected, indicating that mitochondria in some patients haveregular mtDNA transcription and initation of replication activities,whereas in other patients one or both of these activities are reduced orincreased. Thus reduced levels of mtDNA do not necessarily imply reducedmtDNA processing activity in disease, a notion that might be linked tothe extent and/or the progression of the disease.

18A and 18B. Assays on patients diagnosed with progeria-relatedsyndromes, and not diagnosed as mitochondrial diseases. (A) mREPlabelling (red) on primary fibroblasts from patients withprogeria-related syndromes, but that were of diagnosed as mitochondrialdisease or mitochondrial-related diseases. Fibroblasts form healthyindividuals (control) and from a syndrome of sensitivity to UV that isnot associated with progeria are also shown. Nuclei (blue) are labelledby Hoechst Scale bar=10 μm. (B) Fluorescence intensity quantification ofmREP labelling. The red line indicates the value corresponding toaverage controls. T-test compared to controls, (*) p≤0.01; (***)p≤0.001. Note that mREP labelling is reduced or increase in the moderateand severe progeria, respectively. Thus, diseases that are not notidentified for mitochondrial impairment reveal affected mitochondrialfunction by mREP labelling.

FIGS. 19A to 19D. Assays on human cells on long-term treatment with AZT.HeLa cells were treated with AZT from 1 to 15 days. Several parameterswere analysed at the indicated time points: (A) mitochondrial mass byTOM22 immunolabeling; (B) mtDNA content by qPCR; (C) mREP, and (D)mTRANS. T-test compared to untreated controls. A star indicatep<0.01-0.001. Note that after treatment with AZT, in spite of low or novariation in the mitochondrial mass and in mtDNA content, mREP andmTRANS values are dramatically reduced from the first day of treatment.Thus, mREP/mTRANS labelling indicates affected mitochondrial function,even when classical mitochondrial parameters have similar or slightlyaffected values compared to controls.

FIGS. 20A and 20B. Assays on cells treated with rifampicin. HeLa cellswere treated for 24 h with rifampicin at the indicated concentrations.Rifampicin was dissolved in DMSO. Pink columns indicate experimentsperformed in the presence of rifampicin, and green column in thepresence of equivalent amounts of DMSO-containing buffer. White columnsindicate untreated controls. (A) mTRANS and (B) mREP labelling. Notevariations in the levels of mTRANS and mREP at different concentrationsof rifampicin, associated or not with variations in the presence ofbuffer alone. This experiment indicates that treatments with potentialcytotoxic effect (increasing cell mortality was observed with increasingconcentration of rifampicin, not shown) affect mtDNA processing.Alterations of mTRANS and/or mREP values are detected at low doses ofrifampicin.

FIGS. 21A and 21B. Assays on patients diagnosed with cancers. (A)Labelling of isolated cancer cells and normal blood cells. Left panels:labelling of the cytoskeleton (F-Actin, red, by rhodamine-phalloidine)and of the mitochondrial mass (TOM22 immunostaining). Right panels:mTRANS and mREP labelling. (B) Fluorescence intensity quantification ofmTRANS and mREP. Note the higher levels of mTRANS and mREP labelling incancer versus normal cells. However normal cells (from blood) weresmaller in size, and had a especially reduced cytoplamsic mass, comparedto that cancer cells tested here. When different cell types arecompared, values of mTRANS/mREP should therefore be analysed taking intoaccount also the cell size and the mitochondrial mass.

FIG. 22 . Efficiency of the 3D-FISH/mTRIP method of the invention is notaffected by the size of the probe: Upper panel: schematic representationof the position of the probes (with the mitochondrial coordinates) andof the genes present in the region tested; tRNAs are not represented.Mitochondrial coordinates according to MITOMAP(http://www.mitomap.org/MITIMAP). Lower panels: mTRIP FISH labelling ofHeLa cells with the indicated probes (red). Nuclei (blue) are labelledwith Hoechst. Scale bar=10 μm. On the right, fluorescence intensityquantification of 3D-reconstructed cells. n=30; three independentexperiments. T-test, did not show significant differences between thesamples.

FIG. 23 . RT-qPCR and qPCR primers. The sequence of forward and reverseprimers for RT-qPCR (upper panel) and qPCR (lower panel) is indicatedafter the name of the probe that also indicates the gene analysed.Number in parenthesis indicate different sets used to test the samegene. The pair A-B1 amplifies a mtDNA region included in 7S, while thepair A-B2 amplifies a region beyond 7S in the direction of the H-strand.Reference is indicated in the last column.

EXAMPLES A. Materials and Methods

Cells and Culture Conditions.

Human HeLa cells and IMR90 primary fibroblasts (purchased from ATCC)were grown in MEM medium with 10% foetal bovine serum (FBS), HeLa rhoºcells in DMEM medium with 10% FBS 1 mM sodium pyruvate and 0.2 mMuridine, at 37° C. and in the presence of 5% CO2. Cells cultures weresplit at regular intervals for different experiments as required. IMR-90cells were at passage 15. Culture under low oxidative stress weretreated with 50 μM H2O2 for the time indicated.

Reagents and Antibodies.

BrdU, anti-TOM22 Atto488, and Hoechst 33342 were purchased from Sigma;anti-BrdU antibody from BD Biosciences; MitoTracker® Green FM, andsecondary antibodies (Goat anti-mouse antibodies and Goat anti-rabbitantibodies Alexa® Fluor 555 or Alexa® Fluor 488 conjugated) werepurchased from Invitrogen.

Immunofluorescence (IF).

Cells plated on slides were fixed with 2% PFA and permeabilized with0.5% Triton X-100. The slides were incubated in blocking buffer (BSA 5%;PBS 1×) for 1 hr then with the primary antibody for 1 hr. A secondaryanti-mouse or anti-rabbit antibody Alexa® Fluor 555 or Alexa® Fluor 488conjugated was applied. The DNA was stained with 10 μg/ml Hoechst 33342and the image analysis was carried out using Perkin-Elmer Ultraview RSNipkow—spinning disk confocal microscope. For MitoTracker analysis, 200nM MitoTracker® Green FM were added to fixed/permeabilized cells andincubated for 1 hr.

Probe Labeling and Denaturation.

The DNA probes for FISH were labeled by nick translation of PCRproducts, incorporating Atto425-dUTP, or Atto488-dUTP, or Atto550-dUTP,using commercial kit (Atto425/Atto488/Atto550 NT Labeling kit, JenaBioscience). 40 ng of labeled probes were mixed with 400 ng of sonicatedsalmon sperm DNA (Sigma) and hybridization buffer (50% formamide, 10%dextran sulfate, in 2×SSC pH 7.0). The hybridization mix was denaturedat 80° C. for 10 min then kept at 37° C. for 30 min.

Modified 3D-FISH and 3D-FISH Coupled IF.

Cells plated on slides were fixed with 2% PFA and permeabilized with0.5% Triton X100. Cells were then incubated in 50% formamide(pH=7.0)/2×SSC for 30 min at RT, and denaturated in 70% formamide/2×SSCfor 5 min at 75° C. Hybridization was done with 40 ng of probe (singleprobe or mix) for 16 hrs at 37° C. After washing the slides in 2×SSC,1×SSC then 0.1×SSC, the DNA was stained with 10 μg/ml Hoechst 33342, and40 ng of probe (single probe or mix) and the image analysis was carriedout using spinning-disk Perkin Elmer confocal microscope. Experiments atsaturation were performed with 200 ng of probe. When required,fixed/permeabilized cells on slides were treated with RNAseA (100 μg/ml,Roche), or RNAseH (100 U/ml, NEB) or DNAseI (100 U/ml, Invitrogen) for 1hr at 37° C. When more than one nuclease were used, the enzymes wereeither added simultaneously or the second nuclease was added afterincubation with the first nuclease, followed by three washes with PBS,and further incubation for 1 hr at 37° C. For 3D-FISH coupled IF, afterhybridizaton and 0.1×SSC wash, the immunofluorescence procedure wasapplied.

BrdU Incorporation.

Cells plated on slides were incubated for 10 min in the presence of 100μM BrdU, then immediately fixed in 2% PFA (10 min), treated for 10 minwith 4N HCl and 0.5% Triton X-100, and neutralized for 30 min by 100 mMsodium borate. Cells were blocked in 5% BSA in PBS and permeabilizedwith 0.5% Triton X100²⁶. BrdU was detected by immunostaining withanti-BrdU antibody. The DNA was stained with 10 μg/ml Hoechst 33342, andthe image analysis was carried out using spinning-disk Perkin Elmerconfocal microscope.

FISH Coupled BrdU.

Cells plated on slides were fixed with 2% PFA and permeabilized with0.5% Triton X100. Denaturation was performed using buffer containing 10mM Tris HCl pH 8.0, 50 mM KCl, 5% glycerol at 95° C. for 8 min. Theslides were washed in 0.1×SSC and series dehydrated in 70%, 90%, and100% ethanol and finally air-dried²⁷. Hybridization was done overnightat 37° C. After washing the slides in 2×SSC then 0.1×SSC, the slideswere incubated in blocking buffer (BSA 5%; PBS 1×) for 1 hr, thenincubated with mouse anti BrdU antibody for 1 hr. A secondary anti-mouseantibody Alexa® Fluor 555 or Alexa® Fluor 488 conjugated was applied.The DNA was stained with 10 μg/ml Hoechst 33342 and the image analysiswas carried out using spinning-disk Perkin Elmer confocal microscope.

Confocal Acquisition, 3D-Reconstruction and Quantification.

Confocal acquisitions were performed using a spinning-disk Perkin-ElmerUltraview RS Nipkow Disk, an inverted laser-scanning confocal microscopeZeiss Axiovert 200M with an Apochromat 63×/1.4 oil objective and aHamamatsu ORCA II ER camera (Imagopole, PFID, Institut Pasteur). Opticalslices were taken every 200-nm interval along the z-axis covering thewhole depth of the cell, at resolution of 1.024/1.024 pixels.Three-dimensional reconstruction was achieved using the IMARIS software(Bitplane). Fluorescence quantification was done using a single-imagingframe collection and ImageJ 1.34-s software (post-acquisition analysis).The perinuclear location of FISH-labelled organelles corresponds tomitochondria located within 2 |im from the nuclear surface. Thepercentage of perinuclear 3D-FISH mitochondria was calculated on thetotal 3D-FISH labelling. Quantification of mREP-positive andmREP-negative mitochondria was performed on either Polγ or TFAMimmunolabeled areas. For each condition, 300 samples of identicalsurface were analysed. Co-localization studies were done with ImageJJACoP plug-in²⁸.

Statistical Analysis.

The significance of differences between data was determined usingStudent's t test for unpaired observations.

RT-qPCR.

Total RNA was isolated from HeLa cells and IMR90 primary fibroblastsusing a RNAeasy Mini kit (Qiagen) and a RNAeasy Micro kit (Qiagen),respectively. The total RNA was treated with DNaseI (Qiagen), thenreverse-transcribed using Superscript® III Reverse transcriptase(Invitrogen). Real-time quantitative PCR was performed using Power SybrGreen PCR Master Mix (Applied Biosystems) and the rate of dyeincorporation was monitored using the StepOne™ Plus RealTime PCR system(Applied Biosystems). Three biological replicates were used for eachcondition. Data were analyzed by StepOne Plus RT PCR software v2.1 andMicrosoft excel. TBP transcript levels were used for normalisation ofeach target (=ΔCT). Real-time PCR CT values were analyzed using the2^(−ΔΔCt) method to calculate the fold expression (Δ (Δ²CT)method)²⁹.Custom primers were designed using the Primer3Plus online software(http://www.bioinformatics.nl/cgi-bin/primer3plus.cgi). Primers used foramplification are available upon request.

B. Results

Identification of Mitochondrial Subpopulations by Improved FISH (mTRIP)

To gain insight into the dynamics of mitochondrial DNA and RNA insidethe organelle, the inventors have developed a novel approach calledmTRIP (Mitochondrial Transcription and Replication Imaging Protocol)that labels simultaneously DNA and RNA, especially mtDNA and mtRNA inhuman cells, by improving fluorescence in situ hybridization (FISH), andperformed 3D confocal acquisitions (3D-FISH). mTRIP is a combination ofDNA FISH and RNA FISH techniques, and it limits the use of potentiallydamaging agents for macromolecules. Since proteins are not destroyedduring this treatment, and in contrast to existing protocols, 3D-FISHhave been coupled to immunofluorescence (FIG. 1 . D-G). Hence, it waspossible for the first time to monitor, in particular quantativelymonitor, mitochondrial DNA, RNA and proteins simultaneously. Moreover,the intensity of fluorescence could be quantified, thereby permitting arelative assessment of these nucleic acids with single-cell resolution.

TOM22, a subunit of the mitochondrial outer membrane translocase (Yanoet al. 2000) which is uniformly distributed in mitochondria, is usedhere as an indicator of mitochondrial mass. In this context,mitochondria are visualised as individual units or structured in theinterconnected mitochondrial network (FIG. 1 H). Colabelling of mTRIPprobes with TOM22 immunofluorescence (IF) was performed to assess thedistribution of FISH labelling in mitochondria. We observed that mTOT, amixture of 14 probes that cover the entire mitochondrial genome(Table 1) labelled only a fraction of the mitochondrial network in humancells (FIG. 1 I). This labelling with mTOT marked small structureswithin the mitochondrial mass suggesting that they might representnucleoids (see below).

Strikingly, 3D-FISH revealed that the labelling occurred in a distinctfraction of mitochondria, located predominantly in the perinuclearregion in single human HeLa cells (FIG. 1 a; 77.78%±1.72% of labelledmitochondria are located within 2 μm from the nuclear border). Thisresult was intriguing since the inventors used probes that cover theentire 16.5 kbp of the mitochondrial genome (mixture of equimolaramounts of 14 probes, called mTOT, Tables 1-2) and expected allmitochondria to be labelled. Treatment of cells with either DNaseI,RNaseA or RNaseH (specific for DNA-RNA hybrids), and combinations ofthese specific nucleases before hybridization with the mTOT probesshowed that 84% of labelling targets RNA, corresponding to the missingsignal in the presence of RNaseA (FIG. 1 b, c). Moreover, about 23% oflabelling corresponded to DNA and/or structured RNA since it wasresistant to combined RNaseA and RNaseH treatment. These valuescumulated exceed 100% since treatment with some nucleases increases theintensity of labelling, see below. Interestingly, the higher intensityof fluorescence observed in the presence of RNaseH (1.4-fold compared tountreated cells) revealed that removal of the RNA moiety from RNA-DNAhybrids made DNA available for pairing with the fluorescent probe. Thesehybrids probably correspond to transcripts bound to their DNA template.Treatment of samples with RNaseH and subsequently with DNaseI restoredthe fluorescence levels of untreated cells (FIG. 1 c ), confirming thatthe DNA portion of RNA-DNA hybrids paired with the fluorescent probeafter disruption of the RNA moiety. The latter observation, and theapparent absence of effect of the DNaseI treatment, indicated that mtDNAis available in limited amounts for binding with fluorescent probes,unless it is engaged in a local open structure.

Treatment with proteinase K prior to mTRIP resulted in a large increasein the signal (154% for mTOT, 206% for mTRANS and 202% for mREP, FIGS. 1, J and K; the last two probes recognize RNA and DNA, respectively, seebelow) compared to untreated cells, indicating that some mtRNA and mtDNAwere inaccessible to the probes because they could be bound to, ormasked by proteins (FIG. 1 , J-K). In spite of this increase in signalintensity, proteinase K treatment was avoided here because mTRIP wasfrequently coupled to immunofluorescence for the detection of proteins.

TABLE 1 Coordinates of the probes. The start and end points of probesused for FISH experiments are given on the mitochondrial DNA(NC_012920.1, NCBI or GenBank or MITOMAP accession number, was used asreference). Individual probes are indicated in the upper panel. Mix ofmore than one probe and their composition are indicated in individualpanels below. All probes are oriented in the direction of transcriptionof the H strand, with the exception of probe ND6 that is in the inverseorientation (transcription on the L strand). Probe start end size 1 19052866 961 2 2842 3554 712 3 3451 4825 1374 4 4805 6129 1324 5 6032 74201388 6 7400 8518 1118 7 8498 9824 1326 8 9804 11190 1386 9 11107 126181511 10 12513 13517 1004 11 13416 14836 1420 12 14805 16055 1250 1315778 600 1376 14 501 2024 1523 13-1 16034 521 1041 14-1 650 1598 949ND1 3515 3715 200 ATP8 8366 8566 200 ND6 14658 14180 479 mREP 446 544 98PH-1-2 546 746 200 PL-OH 225 425 200 7S 16366 16566 200 mTRANS probes 2,6, 11 mTOT probes 1 to 14 mTOTΔr probes 3 to 13 (rRNA probes excluded)human mt genome size: 16568 bp3D-FISH/mTRIP Labels Transcript Profiles of Mitochondria

To investigate the nature of the mitochondrial subpopulations revealedby this approach, the inventors have performed FISH with each of thesingle 14 probes that were combined in mTOT, in the presence and in theabsence of DNAseI or RNAseA. The inventors have observed that each proberecognized a specific subset of mitochondria and not the entiremitochondrial network (FIG. 2 a ), indicating that not only theintensity but also the distribution of the subset of the labeling variedas a function of the mtDNA region tested. It further indicated that onlya subpopulation of mitochondria carries detectable amounts of the targetnucleic acid, and that mitochondria may not be functionally alike.Saturation experiments indicated that labelling of a subset ofmitochondria was not due to limited concentrations of probe (FIG. 5 ).Treatment with nucleases (DNAseI or RNAseA) showed that all of theprobes recognized essentially RNA targets, with the exception of probes4, 8 and 13, which also recognized DNA (decrease in fluorescencefollowing treatment with DNAseI of 65%, 97% and 47%, respectively).

The fluorescence measurement of each probe, and its decrease aftertreatment with nucleases, revealed that 16S rRNA represents the majortarget of the labelling (probes 1 and 2, FIG. 2 b ). An intense signalwas also observed for ND1 transcript, whose gene is located moredownstream on the H-strand (probe 3, see also FIG. 10 c ). However, thetranscript levels of ND1 were compatible with those of the othermitochondrial mRNAs using a probe specific for this gene (see below,probe ND1, FIG. 6C). Fluorescence labelling distinctly and progressivelydecreased with probes that cover the middle and the end of the H-strand,indicating a reduced amount of signal for late versus early H-strandtranscripts.

Quantitative RT-PCR analysis of single transcripts confirmed that 16S ispresent in a large excess compared to most of the other transcripts(FIG. 6 a ), as expected¹¹, consistent with RNA levels identified bymTRIP thus validating the FISH data. Furthermore, by coupling3D-FISH/mTRIP for each of the 14 mtDNA probes to immunofluorescence withanti-TOM22, a mitochondrial outer membrane marker that identifies theentire mitochondrial population, the inventors have found that not only16S rRNA (probes 1 and 2) is present in a larger proportion ofmitochondria than are the other transcripts, but also that mitochondriacarry larger amounts of this transcript compared to other transcripts(FIG. 7 ).

RT-qPCR confirmed the 3D-FISH/mTRIP result that 12S rRNA is present atsignificantly lower levels than 16S rRNA (FIG. 6 a ). This wasunexpected given that both rRNAs are transcribed from the samepromoters, PH1 and to a lower extent PH2¹² (see scheme in FIG. 2 a ),and that they were reported previously to be produced at similar levelsin vitro¹¹. Although the possibility that the lower signal for 12S rRNAis due to inaccessibility of primers/probes cannot be excluded, thispossibility seems unlikely since 12S rRNA levels were confirmed by twodifferent tests, and using three different target regions (see also FIG.8 . Low levels of 12S RNA were also found in primary fibroblasts (FIG. 2d , FIG. 6 b ). FIG. 2 a also shows that in HeLa cells the distributionof labelled mitochondria varies as a function of the mtDNA regiontested.

Indeed, 16S rRNA is present mainly in mitochondria located in theperinuclear region and in tubular, filamentous mitochondria, whereastranscripts of the last third of the H-strand appear in fragmentedmitochondrial entities, distributed more randomly in the cytoplasm.

Thus, mitochondria cluster around the nucleus during processing of the16SRNA, and they spread to the cellular periphery as RNA processing onthe H-strand terminates. This finding was confirmed by quantitativeanalysis of the perinuclear localization of 3D-FISH labelledmitochondrial populations (72.18±2.28% and 72.35±2.66% of labelledmitochondria were located in the perinuclear region with probes 1 and 2respectively; FIG. 9 ). Moreover, quantitative analysis revealedpreferential perinuclear location (72.87±2.66%) also for mitochondriacarrying abundant transcripts, such as ATP8 (targeted by probe 6). Inaddition, perinuclear mitochondria with probes recognizing mtDNA (seebelow) were observed (80.65±1.54% and 73.37±2.24% for probes 8 and 13,respectively, and 65.47±2.24% for probe 4). In general, the perinuclearlocalization of mitochondria labelled by 3D-FISH/mTRIP is high in HeLacells (>50% of the total mitochondrial population). Although a differentlocalization of mitochondrial populations according to the detected typeof transcript was also observed in IMR-90 primary fibroblasts, noprevalent perinuclear distribution of mitochondria appears in thesecells (30-47%, all probes included; FIG. 2 c and FIG. 9 ). Moreover,mitochondria labelled for various RNAs and DNAs appeared to be morefragmented in IMR-90 fibroblasts than in HeLa cells.

Mitochondrial transcripts exist as processed transcripts of singlegenes, and unprocessed polycistronic transcripts¹. The RNA labellingwhich we observed with 3D-FISH/mTRIP may represent one or both of thesetypes of transcripts. Experiments involving co-labelling with one ormore mtDNA probes helped to distinguish between unprocessed RNAmolecules and individual transcripts (FIG. 10 ). Early transcripts onthe H-strand appeared largely as unprocessed molecules, whereas lateH-strand transcripts appeared frequently as processed molecules.

To analyse further mitochondrial transcription, a new mixture of probeswas used (mTRANS: probes 1, 6, and 11) that are distributed evenly alongthe circular genome. These probes label rRNA and mRNA, and they do notrecognize regions involved in the initiation of DNA replication (seebelow). FISH experiments with mTRANS, in the absence or presence ofnucleases, confirmed that this probe set detects only RNA.

Colocalization of 3D-FISH/mTRIP Labelling with Mitochondrial NucleoidMarkers

The inventors then checked the colocalization between mTRANS that labelsmitochondrial transcripts, and nucleoid markers TFAM and Polγ, whichlabel submitochondrial structures. Extensive colocalization betweenimmunostaining of either Polγ or TFAM and mTRANS (FIG. 3 A) indicatedthat transcripts detected by FISH were mostly confined to labelledmitochondrial nucleoids, as suggested above. Indeed the inventors foundextensive colocalization in all of the combinations tested here,although in each case a fraction of foci did not show colocalization.TFAM and Polγ colocalized with mTRANS by 83.89±4.7% and by 92.69±4.0%,respectively, indicating that less than one tenth of the mitochondrialnucleoids labelled with these markers did not contain transcript levelsdetectable by mTRIP. Conversely, mTRANS colocalized by 63.53±4.1% and by66.23±5.1% with TFAM and Polγ, respectively, indicating that about onethird of the mitochondrial transcripts revealed by mTRIP did not appearto be located in nucleoids where either TFAM or Polγ are present atdetectable levels. The different extents of colocalization among fociare in agreement with significantly different amounts of TFAM innucleoids (Chen, 2005; Shutt, 2010; Spelbrink, 2010; Wai, 2008). In thiscontext TFAM has been recently found to act more as a transcriptionactivator than as a core-component of the transcription machinery invitro (Shutt et al. 2010), and Polγ may be present at low orundetectable levels in transcription-active nucleoids. Therefore, withinthe limits of resolution of mTRIP, mTRANS largely colocalizes withnucleoid markers. The different levels of colocalization between FISHprobes and nucleoid markers might be linked to heterogeneity ofnucleoids (DNA and protein content).

FISH Signal was not Limited by Probes Concentration.

To check whether the intensity labelling by 3D-FISH/mTRIP was limited bythe amount of probes, the inventors have increased by 5-fold the probeconcentration, using 200 ng of mTOT, which corresponds to one of thehighest values described in the literature for FISH experiments³³. Itwas found that increasing probe concentration did not increase theproportion of labelled mitochondria nor the absolute values of thesignal (FIG. 5 ), indicating that the labelling of a subset ofmitochondria by 3D-FISH/mTRIP was not due to limited concentrations ofthe probe.

The inventors have then co-labelled cells with mTOT and with a probethat targets nuclear Alu sequences³⁴ (probe Hs Alu) and found thatlabelling of mitochondrial nucleic acids did not preclude the labellingof nuclear nucleic acids (FIG. 5 ), indicating that the accumulation ofmtDNA labelling in the perinuclear region was not due to inaccessibilityof the probe to the nucleus. This experiment also confirmed thatlabelling with mtDNA probes was specific to mitochondrial nucleic acids.

HeLa cells were also labelled with a further mix of probes, calledmTOTΔr, that includes all probes present in mTOT with the exception ofprobes 1, 2 and 14 that cover the rDNA portion of the mt genome. FIG. 5shows that the intensity of labelling in the absence of rDNA probes wasat least as high as with the probe mTOT, indicating that 3D-FISH labelsmitochondrial mRNAs even in the presence of large amounts of rRNAs. Theinventors have also observed that mTOTΔr-labelled mitochondria were notmostly located in the perinuclear region as it was the case with themTOT mix. This experiment suggested that different combinations of mtDNA probes label distinct mitochondrial populations. It also indicatedthat mTOT-labelled mitochondria located in the perinuclear regionlargely correspond to organelles that contain rRNAs. This notion wasconfirmed by experiments with individual probes (see FIG. 2 a,b ) thatshowed the highest labelling for 16S rRNA.

16S but not 12S rRNAs was Present in Larger Amounts than the OtherTranscripts and was Produced by a Larger Proportion of Mitochondria

12S and 16S rRNA are transcribed in vitro about 10-30 fold more than theother genes on the H-strand³⁵. rRNA transcripts are mostly produced frompromoter PH1 and terminate at specific regions located downstream of 16Swhereas mRNAs and tRNAs are essentially produced from the PH2promoter³⁶, see scheme in FIG. 2 a . By 3D-FISH, the inventors havefound that transcripts containing 16S rRNA (probes 1 and 2) are presentin larger amounts than the other transcripts, as expected, butsurprisingly this was not the case for transcripts containing 12S rRNA(probe 14, FIG. 2 ). This finding was confirmed by quantitative RT-PCR(qRT-PCR) analysis of the 16S and 12S rRNAs (FIG. 6 a ), and by a secondfluorescent probe (14-1) in the region of the 12S RNA (FIG. 8 ). Highlevels of 16S but not of 12S rRNA were observed also in human primaryfibroblasts (FIG. 2 d , FIG. 6 b ).

High levels of fluorescence were observed, surprisingly, also for probe3 that essentially covers the ND1 gene localised downstream of 16S onthe H-strand. Although the signal for probe 3 was lower than for 16SrRNA (probes 1 and 2), as expected³⁵, it was at least two-fold higherthan for the other genes located downstream of rRNA transcriptionterminators (FIG. 2B). The elevated fluorescence with probe 3, observedin HeLa cells but not in primary fibroblasts (FIG. 2 d ), seemed due tothe targeting of unprocessed transcripts that also contain 16S (see nextsection). The intensity of fluorescence labelling dropped by at leastone half with probes 4 to 7 that recognize the downstream region of theH-strand (genes ND2 to the COM), and even more with probes 8 to 12 thatcover the most downstream region, from ND3 to CytB. Interestingly, lowlevels of labelling appeared also for probe 13, that recognizes theregion with the D-loop. On the L-strand, probe ND6 labels the regioncontaining the ND6 gene with intensity comparable to that of probes 4-7on the H-strand. It should be noticed that the fluorescence intensity oftranscripts detected with probes 4, 8, and 13 was even lower than theactual value, given that these probes also recognize DNA (FIG. 2 b ).

The production of large amounts of a given RNA may originate fromelevated transcription by individual mitochondria or from a large numberof mitochondria implicated in transcription, or both. To investigatethis aspect, the inventors have coupled 3D-FISH/mTRIP of each of the 14mtDNA probes to immunofluorescence with anti-TOM22, a mitochondrialouter membrane marker that identifies the entire mitochondrialpopulation. First, the percentage (p) of co-localization betweenanti-TOM22 and each probe was assessed. The inventors have found that alarge proportion of mitochondria (49-69%) was labelled with probes 1 to3, and with probes 6-7, while only 19-38% of mitochondria are labelledwith the remaining probes (FIG. 7 a-b). These data indicated that alarger number of mitochondria carry 16S to ND1 RNAs, and COII to COIIIRNAs, than of the other mitochondrial transcripts. Then, for each probethe intensity of fluorescence present in TOM22-labelled mitochondria wasevaluated, as an indication of the relative amount of transcriptscarried by that mitochondrial population. Therefore, for each probe thepercentage of labelled mitochondria (value p, see above) was multipliedby the intensity of fluorescence of the probe. It was observed thatprobes 1 to 3 had the highest values, whereas values were 2.5-fold lowerfor probes 6-7, and until 20-fold lower for the remaining probes (FIG. 7c ). All together, these results indicated that not only 16S and to aminor extent ND1 RNAs were present in a larger proportion ofmitochondria than were the other transcripts, but also that mitochondriacarried larger amounts of these than of the other transcripts.Conversely, the remaining transcripts were present in a small proportionof mitochondria where they were also present in little amounts. Anintermediary situation was observed for CoII to COIIIRNAs (probes 6-7),that were present in a relatively large portion of mitochondria but insmall amounts therein.

Labelling of Unprocessed and Processed Transcripts

An intriguing result of 3D-FISH experiments concerned the high levels ofRNA labelling for probe 3 that essentially covers ND1 (see above). Highlevels of ND1 labelling may results from PH1 transcription of rRNAs thatdid not stop at terminators or, alternatively, from a particularlylong-lived RNA, although it was not reported that the ND1 transcript wasmore long-lived than the other mRNA in HeLa cells³⁷. In agreement withthe first hypothesis, the levels of ND1 labelling (probe 3) were closeto those of 16S rRNA (probes 1-2). The inventors have reasoned thatlarge amounts of ND1 RNA may result from leaky termination oftranscription from PH1. To check whether ND1 and 16S RNAs labelled by3D-FISH were present on the same molecules and, more in general, whetherRNA labelling by 3D-FISH targeted polycistronic precursor RNAs and/orprocessed transcripts the inventors have performed 3D-FISH with two orthree probes simultaneously. It was found that labelling with probes 2and 3 mostly overlapped (92±1.4% of probe 2 colocalized with probe 3,and 84±1.9% of probe 3 colocalized with probe 2, FIG. 10 ), indicatingthat 16S and ND1 RNAs were essentially located on the same molecule oron distinct molecules that were present at equimolar amounts on the samemitochondrial entities. This was not the case for probe 4 that targetedthe region just downstream of ND1, and that colocalized with probes 2and 3 in only 23±3.2% and 27±3.2% of cases, respectively, FIG. 10 .Thus, if 16S and ND1 RNAs labelled by 3D-FISH were present on the samemolecule, then a relevant part of PH1 derived rRNA transcripts may nothave stopped at termination signals, but proceeded through ND1, at leastin HeLa cells.

The inventors have performed co-labelling with additional pairs ofprobes to verify the simultaneous presence of transcripts inmitochondria. It was found that probe 14, that labelled 12S rRNA presentat the beginning of the H-strand transcript colocalized with probe 12,that labelled CytB present at the end of the same transcript, in 55.6±7%of cases, indicating that mitochondrial entities showing co-localizationeither contained the 5′ and the 3′ end of the PH2-directed transcript,i.e. the complete H-strand transcript, or that 12S and CytB processedtranscripts were present in equimolar amounts on the same mitochondrialentities (FIG. 10 ). It was also observed a large overlap (>60%) forlabelling with probes 7 and 9 that covered close regions localized inthe second half of the H-strand, indicating that most mitochondrialentities contained both transcripts or that these transcripts werepresent on the same molecule. The levels of colocalization decreasedwith the adjacent probes 9 and 10, that additionally showed a ratherdiverse spatial distribution, indicating that these probes mostlylabelled transcripts located on different mitochondrial entities or ondifferent molecules (FIG. 10 ). These results are summarized in FIG. 10f that takes into account the percent of co-localization and the totalfluorescence intensity of the tested probe. Although just indicative inquantitative terms, these data nevertheless confirms a relevantco-localization of transcripts targeted by probes 2 and 3 (located inthe first quarter of the H-strand), and also by probes 7 and 9 (locatedin the third quarter of the H-strand). On the contrary, data show ascarce co-localization of transcripts targeted by probes 2-3 and 4, andprobes 9 and 10, revealing that most of these transcript pairs were notpresent in the same mitochondria. In conclusion, colocalizationexperiments strongly suggested that transcripts labelled by 3H-FISHprobes represented both unprocessed RNA molecules, in particular theearly transcripts on the H-strand, and processed individual transcripts.This proof of principle showed that processed and unprocessed RNAmolecules could be identified for any mitochondrial gene of interestusing, appropriate pairs of probes in co-localization experiments.

qPCR Analysis of Mitochondrial Transcripts or Correlation Between3D-FISH/mTRIP and RT-qPCR Transcript Levels

To check whether the proportion of the various transcripts detected with3D-FISH in distinct mitochondrial populations were consistent with thetranscript levels of the mitochondria, the inventors have performedqRT-PCR experiments for each mitochondrial rRNA and mRNA gene (FIG. 6 a). A direct comparison of 3D-FISH and qRT-PCR data was not suitablesince FISH probes used here cover regions larger than a single gene,with the exception of probes 5 and 10 that cover only COI and ND5,respectively. Nevertheless, quantitative RT-PCR analysis of the othermitochondrial genes analysed showed expression level profiles compatiblewith those observed by FISH analysis. The transcript levels of ND1 werecompatible with FISH using a probe specific for this gene (probe ND1,FIG. 6C) but not with the longer probe 3, which probably labels alsounterminated rRNA transcripts (as suggested in the previous section“Labelling of unprocessed and processed transcripts”). In conclusion, agood correlation between RT-qPCR, which detects transcripts of theentire mitochondrial and cellular populations and mTRIP, which revealsRNAs in a fraction of mitochondria and at the single-cell level, wasnoted.

TABLE 2 Position of the probe on the human mitochondrial genome. Thecoordinates of the genetic element present at a given position of themitochondrial genome (NC_012920.1, NCBI or GenBank or MITOMAP accessionnumber) are indicated in column 1 (data from MITOMAP:http://www.mitomap.org/MITOMAP/HumanMitoSeq). The name of the elementitself is indicated either on column 2 or 3 (direct and inverseorientation with respect to the direction of transcription of theH-strand, respectively). In the last three columns is/are indicated theprobe(s) that hybridize with the indicated region. Even hybridization ofa few nucleotides is indicated. position element element probe probeprobe 110-441 Origin H 13 13-1 213-235 CSB1 13 13-1  299-31 5 CSB2 1313-1 346-363 CSB3 13 13-1 392-445 PL (or LSP) 13 13-1 545-567 PH1 14 1313-1 577-647 tRNAphe 14 13 645 PH2 14  648-1601 12S RNA 14 1602-1670tRNAval 14 1671-3229 16s RNA 14 1 2 3230-3304 tRNAleu (UUR) 2 3307-4262ND1 3 2 4263-4331 tRNAile 3 4365-4400 tRNAgln 3 4402-4469 tRNAf-met 34470-5511 ND2 4 3 5512-5579 tRNAtrp 4 5587-5655 tRNAala 4 5657-5729tRNAasn 4 5721-5755 Origin L 4 5761-5826 tRNAcys 4 5826-5891 tRNAlys 45904-7745 COI 4 5 6 7446-7514 tRNAser (UCN) 6 7518-7585 tRNAasp 67586-8329 con 6 8295-8364 IRNAlys 6 8366-8572 ATP8 7 6 8527-9207 ATP6 79027-9990 COIN 7 8  9991-10038 tRNAglu 8 10059-10404 ND3 8 10405-10469tRNAarg 8 10470-10766 ND4L 8 10760-12137 ND4 9 8 12138-12206 tRNAhis 912207-12265 tRNAser(AGY) 9 12266-12336 tRNAleu(CUN) 9 12337-14148 NAD5 910 11 14149-14673 NAD6 11 14674-14742 tRNAglu 12 11 14747-15887 CytB 1213 15888-15953 tRNAthr 12 13 15956-16023 tRNApro 12 13 13-1 16024-191 7SDNA 12 13 13-13D-FISH/mTRIP Revealed mtDNA Initiation of Replication

The inventors have observed above (FIG. 2 b ) that three probes (13, 4and 8) detected not only RNA but also DNA. Interestingly, probes 13 and4 included the regions of initiation of replication of the H- and theL-strand, respectively, suggesting that these probes detected DNAregions engaged in the initiation of replication. Probe 8 included theND4 region, where an additional origin of replication for the L-strandhas been observed using atomic force microscopy¹³ and which is expectedto be activated less frequently than the two major ones. These threeorigins are located almost symmetrically on the mt genome, asschematized in FIG. 3 a . To assess whether DNA labeling by3D-FISH/mTRIP was associated with initiation of DNA replication, theinventors have investigated the region covered by probe 13 which isunique within the entire mitochondrial genome, as it contains a sequencethat is not transcribed, according to the terms used herein, inparticular a sequence that is substantially not transcribed, andtherefore it should be present only in its DNA form (FIG. 3 b ). Theinventors generated a second probe (13-1) which covered a shorter regionthan probe 13 and which did not contain genes, and a third probe (mREP)that covered only non-transcribed DNA (FIG. 3 b ). 3D-FISH/mTRIP witheach of the three probes showed that only mREP resulted in fullyDNaseI-sensitive and fully RNaseA-resistant labelling (FIG. 3 c , FIG.11 ) indicating that this probe specifically labelled DNA.

Since the DNA region labelled by mREP is normally present in the genomeof all mitochondria, the inventors have reasoned that 3D-FISH/mTRIPlabelled only mitochondria where this DNA region was structurallyaccessible, because of initiation of DNA replication (O_(H) origin)nearby. To assess whether this was the case, immunostaining of DNApolymerase γ (Polγ), the enzyme responsible for replication mtDNA, wascoupled to 3D-FISH/mTRIP. mREP is associated with nucleoids that containfactors involved in DNA replication and transcription. mREP labellingcoupled to immunofluorescence with Polγ or TFAM showed that this was thecase (74.4±2.5% colocalization of mREP with Polγ, and 71.7±1.5% withTFAM; FIGS. 3D-E), and also that most of the mREP foci werepreferentially localized to Polγ and TFAM rich areas (FIG. 3D-E).Moreover, it was found that the intensity of fluorescence of Polγ almostdoubled in areas labelled with mREP compared to mREP-negative areas(cells n=30; mt areas n=300, FIG. 3 d ). Furthermore, mREP-positivemitochondria were associated with higher levels of TFAM immunolabellingcompared to mREP-negative mitochondria (FIG. 3 e ). TFAM is a proteinimplicated both in transcription of mtDNA and in binding to the mtDNA,and whose levels are correlated with increased mtDNA¹⁴.

mREP Labelling Precedes the Increase of mtDNA Content

The inventors have reasoned that if mREP labelling is an indicator ofmtDNA initiation of replication, it should anticipate the increase inmtDNA content. To assess whether is was the case, HeLa cells weretreated in culture with low doses (50 μM) of H₂O₂, known to increase themtDNA copy number and the mitochondrial mass³⁸. As expected, treatmentwith H2O2 resulted in an increase of about 30% of the mitochondrialmass, measured by the intensity of fluorescence of the mitochondrialprotein TOM22, and in the increased expression of the mitochondrialbiogenesis marker Nrf1 (FIG. 12 a,b). It was observed that mREPlabelling increased by about 70% 1 h after treatment, when the DNAcontent was low probably due to the stress of the treatment, andreturned to control values 3 h after treatment when the originalmitochondrial DNA content was restored. At 24 h, at high mtDNA content,mREP labelling was as low as in untreated cells. Thus, mREP labellingincreased when the mtDNA content was low and returned to original valueswhen the mtDNA content was elevated. Moreover, mREP labelling wasfollowed by a rise in the mtDNA content within 2 hours, compatibly withthe time necessary to replicate the mt genome (about 92 minutes fortotal mtDNA replication³⁹), supporting the notion that mREP detected theinitiation of mtDNA replication. Under low doses of H₂O₂ we alsoobserved an increase of mitochondrial but not nuclear transcription,measured by labelling with the mTRANS probe and CytC expression,respectively.

Furthermore, the inventors have checked BrdU incorporation (10 μM BrdUfor 24 h), an indicator of DNA replication, in mitochondria. It wasfound that mREP-positive entities co-labelled with BrdU, confirming thatmREP labelled mitochondria engaged in DNA replication. Importantly, mREPlabelled only a subset of BrdU-positive mitochondria, indicating thatmREP did not detect extensive or completed replication of the completemt genome but rather a special event corresponding to initiation of DNAreplication. All together these data support the notion that mREP is asa marker of the initiation of mtDNA replication.

In this context, the intensity of BrdU labelling was lower inmREP-positive compared to mREP-negative areas, in agreement with thelimited incorporation of a nucleotide analogue at the beginning ofreplication of the mitochondrial genome. Taken together, these results,and the unique characteristics of the region of the mtDNA recognized bymREP, support the notion that mREP marks initiation of replication.

Whether DNA synthesis proceeds from O_(H) until the end of the H-strand,or terminates earlier, leading to the formation of the 7S strand andthereby of the D-loop, was not resolved by FISH labelling alone. Toassess whether mREP signal indeed corresponds to the labelling of mtDNAor of 7S DNA, or both, the inventors compared endogenous levels of theseDNAs by real-time qPCR, as described previously (Antes et al. 2010), inuntreated cells and in cells treated with low levels of H₂O₂, addescribed above. The inventors found that the variations observed in themtDNA content after exposure to H₂O₂ and associated with changes in mREPlevels (previously evaluated in the 12S region of the mtDNA arecompatible with variations of the mtDNA and not of 7S DNA, which levelskeep increasing after the mREP signal returns to normal 3 h aftertreatment. Thus, although mREP may label both the productive and theabortive initiation of mtDNA replication (formation of the D-loop),variations in mREP are compatible with productive replication of themtDNA rather than with the formation of the D-loop.

Only a Fraction of Mitochondria are Engaged in Initiation of DNAReplication and/or in Transcription Detected by mTRIP

To assess the fraction and the distribution of mtDNA processingactivities (i.e. mtDNA transcription and replication) within themitochondrial network the inventors performed colabelling with mREP,mTRANS, and TOM22. Notably, 58.9±2.7% and 12.9%±1.3% of themitochondrial mass (TOM22 immunolabelling) colabelled with mTRANS andmREP respectively. Therefore, a significant fraction of the mitochondriawere not labelled with either probe indicating that either they are notinvolved in the transcription of the tested genes and/or in thereplication of mtDNA, or that the levels of the target molecules are notdetectable with this approach. In addition, 71.3±2.9% of foci labelledby mREP also carried mTRANS transcripts whereas only 8.5±0.8% of focicarrying mTRANS were also mREP-positive. These results reveal that themajority of mitochondria involved in the initiation of replication alsocarried mTRANS transcripts, whereas only a minority of mitochondria thatcarried detectable transcripts were also involved in initiation of mtDNAreplication in these cells.

Heterogeneous Labelling of the Regulatory D-Loop Region in Mitochondriawithin Single Cells

The inventors reasoned that if mTRIP can identify distinct mitochondrialpopulations within single cells according to the DNA engaged ininitiation of replication and to the transcript content, it should alsoidentify mitochondria with distinct RNA and DNA labelling patterns inthe regulatory region, which may be fonctional to the regulation ofmtDNA itself. To assess this point, the inventors performed FISH withthree probes located in the D-loop region (probes PL-OH and 7S) and atpromoters of the H-strand (P_(H1) and P_(H2); probe PH1-2), FIG. 15B.They performed single labelling and colocalization experiments with twoprobes in the presence and in the absence of nucleases. Quantitativeanalyses of fluorescence showed that probe PL-OH, located downstream ofmREP in the direction of DNA replication, and which includes the P_(L)and the O_(H) regions, labels accessible DNA structures(DNaseI-sensitive labelling, FIGS. 15 A and C), which might be part ofthe P_(L) transcription bubble, or of the O_(H) replication bubble, orof both. This last possibility is in agreement with a large set ofevidences indicating that transcription from P_(L) is coupled toH-strand replication (Scarpulla 2008).

Probe PL-OH also labels RNA in RNA/DNA hybrids (reduction of labellingin the presence of RNaseH, FIGS. 15 A and C). These RNA molecules mightconsist of R-loops, i.e. the RNA primers for DNA synthesis for the O_(H)origin (Brown et al. 2008), present as processed as well as unprocessedmolecules of various lengths, or regular L-strand transcripts, or both.Interestingly, simultaneous treatment with DNAseI and RNaseH resulted inresidual labelling (19.89%±1.4%) that disappeared when cell were alsotreated with RNAseA (FIG. 15A). This experiment indicates that probePL-OH also labels RNAseH-resistant RNA/DNA hybrids, which RNA becomesaccessible to RNaseA after the DNA moiety is removed by the action ofDNaseI. This finding is in agreement with the notion that the structureof the RNA/DNA hybrids in R-loops may confer resistance to RNaseH (Brownet al. 2008). An increase in the intensity of labelling in the presenceof RNaseA indicates that not only RNA is not a significant target ofPL-OH but also that RNA molecules to a certain extent inhibit thelabelling of the other targets.

More detailed information on the heterogeneity of the nucleic acidscomposition of the D-loop region in mitochondria was provided by thedirect observation of foci (FIG. 15 C). Indeed, PL-OH labelling consistsof small foci with poor fluorescence intensity as well as of large fociof intense fluorescence, and distinct colocalization patterns with theother probes. The large PL-OH foci, which are mostly DNaseI-resistantand RNaseH-sensitive (FIG. 15 A), essentially colocalize with foci ofthe downstream 7S probe located within the 7S region (FIG. 15 A, lowerpanels). Differently from PL-OH, however, large 7S foci are essentiallyRNaseH-resistant. Thus, PL-OH and 7S large foci colocalize but theformer probe recognizes RNA/DNA hybrids whereas the latter recognizesRNA. This pattern is compatible with the labelling of transcripts boundto the DNA template proximally to the promoter (probe PL-OH), and ofsingle-strand RNA distally from the promoter (probe 7S, FIG. 15 C),although the limits of resolution of FISH do not define whether labellednucleic acids are present on the same molecules. In addition to thisprevalent type of labelling, large DNaseI-resistant 7S foci andapparently DNaseI-sensitive PL-OH foci were detected (pattern 3 in FIG.15 C), which are compatible with the decreased PL-OH signal in thepresence of DNaseI. These foci reveal accessible DNA structure at thelevel of PL-OH whereas RNA is present at the level of 7S. Moreover,RNaseH-resistant PL-OH and 7S foci were observed (pattern 5 in FIG. 15C), which consist either of RNAseH-resistant RNA/DNA hybrids at one orboth loci or of DNA (PL-OH) and RNA (7S). The heterogeneity of mTRIPlabelling in this region within single cells is summarized in FIG. 15D.

Since treatment with RNaseA did not reduce the labelling with probe 7Sto background levels, this probe likely binds other targets than justRNA. Indeed simultaneous treatment with RNAseH and RNaseA resulted insignificant decrease of the signal, compared to RNaseA alone (p=0.0013),indicating that some RNA/DNA hybrids were recognized by probe 7S (FIG.15A).

Interestingly, large PL-OH and 7S foci which were greatly reduced innumber after treatment with RNaseA alone, essentially disappeared aftertreatment with RNaseA and RNaseH (FIG. 4C), indicating that RNA isessential to the structures identified by these foci, and that PL-OH and7S foci are linked. Moreover, large PL-OH and 7S foci disappeared aftersimultaneous treatment with RNAseH and DNaseI whereas treatment withDNaseI had essentially no effect, and treatment with RNaseH affectedonly PL-OH foci. This experiment indicates that the RNA and the DNAmoieties of RNA/DNA hybrids, at least at the level of 7S, are essentialto the formation not only of the structures labelled by probe 7S butalso of the structures labelled by probe PL-OH, further supporting thenotion that the nucleic acids labeled by the two probes are linked.

Interestingly, in the presence of two nucleases, the disappearance oflarge PL-OH and 7S foci was replaced by the appearance of small foci(RNaseA and RNaseH) or of foci of reduced size (DNAseI and RNAseH),which displayed some colocalization (FIG. 15D). However, differentlyfrom large foci, most other foci did not colocalize indicating that inthese cases the nucleic acids labelled by the two probes are not linked.

Importantly, only a limited fraction of mitochondria were labelled withprobes PL-OH and 7S (colabelling with mTOT, not shown), indicating thatin this regulatory region nucleic acids were not accessible or wereaccessible below detectable levels in non-labelled mitochondria.Finally, colocalization experiments revealed that not only PL-OHcolocalizes by 99.44%±0.05% with 7S, thereby further supporting thenotion of a link between the nucleic acids labelled by the two probes,but also that mREP colocalized with 7S by 99.33%±0.07%. This resultindicates that mREP, PL-OH and 7S likely label linked althoughheterogeneous nucleic acid structure(s). Conversely, the majority butnot the totality of 7S colocalizes with mREP (59.8%±2.6%), and withPL-OH (69.7%±2.7%), indicating that 7S also labels RNA that is notinvolved in the structure linked to initiation of replication,compatibly with labelling of L-strand transcripts. In agreement withthis notion, the intensity of labelling with PL-OH was 2.3-fold higherthan with mREP, compatibly with PL-OH targeting not only DNA in thereplication bubble but also transcripts.

In conclusion, probes PL-OH and 7S, identify in a fraction ofmitochondria a variety of structures with distinct nucleic acidcomposition that appear associated with O_(H) DNA replication andL-strand transcription and that coexist in single cells.

mtDNA Transcription Dynamics in the P_(H) Promoters Region at the SingleCell Level

On the other side of mREP, probe PH1-2, which is located in the regionof promoters P_(H1) and P_(H2) (FIG. 15B) is completely sensitive toDNaseI, indicating that it labels essentially DNA (FIG. 15 E). Theaccessible DNA in this region might result from its opening as aconsequence of the nearby O_(H) replication bubble, or represent theP_(H1)/P_(H2) transcription bubble. The overwhelming colocalizationbetween probe PH1-2 and probe 1, which labels 16S RNA transcribedessentially from P_(H1), (FIG. 4 E) supports the second notion.Moreover, the elevated intensity of fluorescence observed with probePH1-2, which is compatible with the signal detected with probe 1 (FIG. 4F), indicates that probe PH1-2 labels DNA in the transcription bubbleformed at P_(H1) for the aboundant transcription of rRNAs. Treatmentwith either RNaseA or RNaseH does not significantly alter the efficiencyof labelling, although foci have a different aspect compared tountreated controls, indicating that although RNA and RNA/DNA hybrids donot appear to bind this probe, they might affect the structure of theDNA to which the probe binds.

A large difference in the extent of labelling among probes located inthe regulatory region of mtDNA was noted. PH1-2 fluorescence intensitywas 6.9-fold higher than mREP, and probe 1 fluorescence intensity, whichmarks the 16S transcript was 10-fold higher, compatibly with robusttranscription of rRNAs (FIG. 15 F). Simultaneous labelling with twoprobes reveal that 48.73%±3.44% of mREP foci colocalize with PH1-2 foci,indicating that although in certain cases accessible DNAs in these tworegulatory regions may be linked, in other cases it is not, compatiblywith the notion that transcription of rRNA can be uncoupled from theformation of the DNA structure that promotes replication in the O_(H)region.

These data are consistent qualitatively and quantitatively (intensity offluorescence, FIG. 15 F) with the expected replication and transcriptionactivities of the regions examined (Chang and Clayton 1985; Clayton1991; Falkenberg et al. 2007; Scarpulla 2008). These findings alsoprovide novel information on concomitant DNA replication andtranscription activities in the regulatory region of mtDNA in singlecells.

C. Discussion

Understanding the dynamics of DNA transcription and replication withinthe mitochondrial network is essential to assess mitochondrial function.Mitochondria appear to be homogeneous as a population within singlecells, although functional differences have been described for synapticand non-synaptic mitochondria in neurons²⁰. The inventors have devisedhere a novel 3D-FISH approach which identifies a variety ofmitochondrial populations in single-cells. These populations differ inthe intracellular localization, in the relative amount of transcriptthat they carry and in their engagement in initiation of DNA replicationand in the signal of the regulatory region of mtDNA, indicating thatmitochondria are more heterogeneous than previously thought in DNAprocessing activities.

The novel FISH protocol (mTRIP) described herein identifies a unexpectedvariety of mitochondrial populations with distinct properties withinsingle-cells. These populations differ in their intracellularlocalisation, in the relative amount of transcripts that they express,in the initiation of DNA replication, and in the signal of theregulatory region of mtDNA indicating that mitochondria exhibit agreater level of heterogeneity in DNA processing activities thanreported previously, including mitochondrial dynamics during mtDNAsynthesis (Davis and Clayton 1996). Only 16S rRNA appears to label mostof mitochondria, and this with an elevated signal per unit, but allother probes identify distinct and occasionally minor mitochondrialfractions. Within the limits of resolution of this approach, labellingof mtDNAs and RNAs was also shown to be correlated with nucleoids, themitochondrial substructures involved in mtDNA processing. The inventorsobserved different levels of colocalization between FISH and nucleoidmarkers, in agreement with the different amounts of regulatory proteinsfound in nucleoids and which might have regulatory functions (Chen andButow 2005; Spelbrink 2010; Shutt et al. 2011).

The 3D-FISH method described herein detected mitochondria andmitochondrial substructures rich in a given transcript, that was presentas a processed molecule, a polycistronic RNA, or bound to the DNAtemplate, the latter likely resulting from ongoing transcription.Moreover, RNAseH treatment revealed the presence of another class oftranscripts (accounting for 38% of additional signal) that were stillbound to the DNA template and likely resulted from ongoingtranscription. The variety of RNA molecules labelled by 3D-FISH, whichalso included truncated and misprocessed transcripts, provided moreextensive information compared to full-length transcripts detected byRT-qPCR. Moreover, the 3D-FISH method described herein permitted thedetailed investigation of mtDNA dynamics, since it labelled relevantmitochondria in single-cells, whereas RT-qPCR only assessed thetranscript levels of entire mitochondrial and cellular populations.

In general, the inventors found a good correlation between RNA levelsdetected with mTRIP and RT-qPCR, thus validating the FISH approachdescribed herein which allows assessing mitochondrial transcripts withinthe mitochondrial network in individual cells.

With these novel tools the inventors have found that, unexpectedly, ofthe two rRNAs produced from the same PH1 promoter¹¹, 16S, but to alesser extent 12S, is abundant in mitochondria. RT-qPCR data confirmedthe 3D-FISH finding, and this was the case in both HeLa cells andprimary fibroblasts. Importantly, variable levels of 16S versus 12S rRNAwere detected in liver cells²¹ and, in their adenylated form, 16S RNAwas more abundant than 12S RNA in the skeletal muscle²², indicating thatlower levels of 12S versus 16S RNA detected with the present analysis,represented a physiological situation.

Importantly, it was observed that mtDNA processing was not alike in allcell types. It was found that in HeLa cells mitochondria carryingabundant transcripts (16S, ND1 and and to a certain extent ATP8) weremainly located in the perinuclear region, whereas the less abundanttranscripts of the last third of the H-strand appeared progressivelydistributed in the cytoplasm and in more fragmented mitochondrialentities. The perinuclear localisation of mitochondria may be requiredfor the nuclear uptake of molecules necessary for intensivemitochondrial transcription and/or DNA replication, or for bufferingCa²⁺ fluctuations from the cytoplasm²³. However, perinuclearlocalization of mitochondria has been also described in cells ofpatients with myopathic and neurodegenerative diseases characterized bymitochondrial dysfunctions^(24,25). In this context, it was interestingto note that perinuclear distribution of most mitochondria, and inparticular of the organelles that produce the predominant 16S RNA, wasnot observed in primary fibroblasts, thus raising the possibility thatsuch localization is associated with mitochondrial impairment.Additional differences characterize mitochondrial DNA in primary cellsversus cancer-derives cell lines. The 3D-FISH method described hereindetected high levels of ND1 RNA in HeLa cells but not in primaryfibroblasts. This transcript was present probably as polycistronic RNAconsequent to leaky termination from promoter PH1, indicating thatmitochondrial rRNA transcription termination may be altered in HeLacells.

Labelling of DNA by the 3D-FISH method described herein appears limitedto locally open structures, as in transcription complexes afterdisruption of the RNA moiety, and in DNA engaged in initiation ofreplication. Interestingly, a third mitochondrial replication originpreviously detected with atom force microscopy and expected to beactivated only occasionally¹³ was revealed in the experiments that wereconducted and its position in the mitochondrial genome defined at ahigher resolution. To date, identification of mitochondrial initiationof replication in single cells has been elusive. Importantly, theinventors have defined the characteristics necessary for a probe tospecifically mark the initiation of DNA replication, and proposed aspecific probe, mREP, which is an efficient marker of initiation ofmtDNA replication. Mitochondria engaged in DNA replication couldtherefore be detected and analysed in cells and under experimentalconditions of biological relevance.

The combination of mtDNA transcription and initiation of replicationlabelling can provide information on mitochondrial dynamics in a varietyof physiological processes. (e.g., the dynamics of mitochondrial DNAtranscription and replication during the cell cycle, Chatre & Ricchetti,in preparation). Moreover, the 3D-FISH method which is described hereincan provide novel information on alterations of mtDNA dynamics andrepresents a novel tool which can impact on disease screening related tothe mitochondrial function.

Investigation of mtDNA regulatory regions by mTRIP identified DNA, RNA,and RNA-DNA hybrids at the expected locations according to currentknowledge on global mitochondrial populations (Chang and Clayton 1985;Clayton 1991; Falkenberg et al. 2007; Scarpulla 2008), thus furthervalidating this approach, which however operates at the single-celllevel. Indeed colocalization between probes pairs reveals structuresthat contain accessible DNA upstream of the replication origin andcomprising the promoters P_(L) on one side and P_(H1) and P_(H2) on theother side. Conversely, RNA is the almost exclusive target in the 16Sregion, as expected for the P_(H1) transcript, and in the 7S regionwhere it probably represents the L-strand transcript. RNA/DNA hybridsare detected at the level of the P_(L) promoter compatibly with theformation of R-loops, that provide the RNA primers for DNA replication,and also at a minor extent at the level of 7S where they mat representL-transcripts bound to the DNA template. Moreover, the intensity ofcolocalization among probes reveals comparable levels of labelling forthe region mREP, that according to our experiments indicates initiationof replication, and the region that that comprises the O_(H) replicationand the downstream 7S transcript, in agreement with the notion thatL-strand transcription and replication are coupled. Our data suggestthat these two processes are not only temporally but also quantitativelylinked. In addition to these aspects, our findings provide novelinformation on the dynamics of the key regulatory regions of the mtDNAwithin the mitochondrial populations.

First, accessible DNA in the O_(H) replication origin and L-strandpromoter may be linked to accessible DNA in the H-strand promoters(about one half of the relative signals colocalize), whereas the twotypes of events appear uncoupled for the remaining half of the signal,indicating that in these cases rRNA transcription is not linked to O_(H)replication/L-strand transcription. Colocalization between these fociand mREP foci (mREP is located in the middle of the opposite promotersP_(L) and PH₁₋₂, and also upstream of the main replication origin O_(H))suggest that rather than being a passive region, mREP appears as anindicator or a key regulator region not only of the main replicationorigin, but also of transcription of both the H- and the L-strands.

Furthermore, mTRIP identifies within a single-cell a variety oflabellings that include a prevalent pattern, and also distinct patternsthat show either higher levels of accessible DNA at the level of theorigin of replication, or RNAseH resistant structures at the level ofthe origin of replication, or else DNA labelled at the level of thetranscription of 7S, compatibly with the activities expected at thesefoci.

In conclusion, by mTRIP the dynamics of mtDNA transcription andinitiation of replication are exposed with unprecedented resolution atthe single-cell level, which may help in further elucidating the linkbetween mitochondrial transcription and replication, and which may beused for future investigations of mtDNA processing under physiologicaland pathological conditions.

D. Conclusion

Mitochondrial DNA (mtDNA) replication and transcription are crucial forcell function, but these processes are poorly understood at thesingle-cell level. By modified fluorescence in situ hybridization,called 3D-FISH, the inventors have identified mitochondria engaged ininitiation of DNA replication in human cells. Mitochondria were alsodistinctly marked according to transcription profiles. Thus, theinventors have documented the existence of mitochondrial subpopulationsin single cells according to the prevalent mtDNA processing activity,indicating that mitochondria may not be functionally alike. Importantly,the inventors have proposed an in situ hybridization procedure, and moreparticularly a 3D-FISH protocol that can be coupled toimmunofluorescence, and they were thus able for the first time tomonitor mtDNA, mtRNA and proteins simultaneously in single cells anddemonstrate significant heterogeneities that have been previouslymissed. With this approach, novel information can be provided on thedynamics of mtDNA processing during physiological and pathologicalprocesses. These findings have implications for the optimization ofdiagnostic tools for mitochondrial diseases, in particular thoseinvolving mtDNA depletion and mtDNA loss.

Since currently available tools including recent improvements⁷, cannotidentify mitochondria engaged in DNA replication, they cannotdiscriminate the transcription profiles of organelles in single cells.Moreover, although sequential RNA and DNA labelling⁸, as well labellingof either RNA or DNA, and proteins^(9, 10) have been performed,immunofluorescence was not directly coupled to FISH to simultaneouslydetect proteins and mitochondrial DNA and RNA. Thus, proteins ofinterest could not be monitored during mtDNA transcription andreplication. As a consequence, it remained unclear how mtDNA processingis coordinated among the many organelles present in each cell andwhether this process is deregulated in subpopulations during disease.Using a novel approach, the inventors have identified mitochondrialsubpopulations engaged in the initiation of mtDNA replication and in RNAprocessing, and assessed their dynamics in single cells. Theses findingsrevealed significant heterogeneities within single cells that have beenmissed previously, and this can impact on how mitochondrial functionsare assessed. Mitochondria with altered processing of DNA and RNA, as indiseases involving mtDNA loss, can be identified with this novelapproach.

E. Applications

The present invention is of particular interest for analyzing theprocessing of DNA, RNA or metabolites in cell(s) or tissue(s), and/oranalyzing the dynamics of said cell(s) or tissue(s), and/or detectingspecific diseases.

As stated above, mitochondrial misfunction is associated with a varietyof diseases (cancers, myopathies, neuropathologies, infections), andwith the ageing process, and can be found in a number of mitochondrialdiseases.

Mitochondrial diseases are diagnosed in 11.5/100 000 adults and childrenper year in the world (˜800 000 patients/year), and 1/4 000 (25/100 000)USA children.

Mitochondrial diseases are difficult to diagnose. Referral to anappropriate research center is critical. If experienced physicians areinvolved, however, diagnoses can be made through a combination ofclinical observations, laboratory evaluation, cerebral imaging, andmuscle biopsies. Despite these advances, many cases do not receive aspecific diagnosis.

Most hospitals do not have a metabolic laboratory and therefore can runonly the most basic tests. In addition, a single blood or urine lab testwith normal results does not rule out a mitochondrial disease. This istrue for organic acids, lactic acid, carnitine analysis and amino acidanalysis. Even muscle biopsies are not 100% accurate.

To date, most of the studies on mitochondria are based on molecularbiology assays (PCR, qPCR, Southern blot), biochemistry (Western blot,ATP/Reactive Oxygen Species (ROS)/membrane potential detection assays),and electron microscopy (for the mitochondrial ultrastructure).

For example, current diagnostic tools for the mitochondrial diseasesencompass Metabolic Screening in Blood and Urine (complete blood count,lactate, pyruvate, plasma amino acids, liver enzymes, ammonia, urineorganic acids . . . ), Metabolic Screening in Spinal fluid (lactate,pyruvate, amino acids, cell count, glucose, protein), Characterizationof Systemic Involvement (echocardiogram, ophthalmologic exam, brain MRI,electrocardiogram, audiology testing), Clinical Neurogenetics Evaluation(karyotype, child neurology consultation, fragile X test, geneticsconsultation).

There is therefore a need for simple, reliable and fast methods andtools for including in diagnosis protocols of mitochondrial diseases andmitochondrial dysfunctions.

In addition, fluorescence imaging tracks separately mitochondrial DNA(mtDNA), mitochondrial RNA (mtRNA), by fluorescence in situhybridization (FISH), and proteins, by immunofluorescence (IF), in fixedcells. However, two aspects restrain the potency of fluorescence imagingof mitochondria. First, even using a combination of different imagingprocedures (for instance IF and RNA FISH, or IF and DNA FISH, of RNA andDNA FISH), it is not possible to detect in the same cell DNA, RNA andproteins. This can be due to cross-reaction of chemicals and damages ofthe samples during the procedure(s). For example, when IF and FISH arecombined, FISH provokes damages to the proteins resulting in a reducedfluorescence signal for the proteins that cannot be interpretedcorrectly. Second, the prior art FISH procedure for the detection ofmtDNA contains large DNA probes (i.e. more than 3 kbp), which generatehigh levels of a specific staining and thus decrease the overallresolution.

Therefore, the development of a novel FISH labeling approach of cellsthat allows the tracking of mitochondrial DNA initiation of replicationat the single-cell resolution is of particular interest to revealdysfunctions at this level. In addition, the present invention furtherallows the simultaneous detection of mitochondrial RNA, and thus themonitoring of transcription events. Bi-dimensional or three-dimensionalimaging can also be performed. Moreover, since the developed FISHprocedure does not damage the epitope/antigen, it permits also thesimultaneous analysis of mitochondrial and/or cellular proteins. In its3D version, this technique has been called called 3D-Fluorescence InSitu Hybridization coupled ImmunoFluorescence (3D-FISH coupled IF) andresults in a drastic modification of the classic FISH procedure in termof cell fixation, permeabilization, mtDNA probes design, size andfluorescence labeling, cell and DNA probes denaturation.

Moreover, although the depletion of mitochondrial DNA is currentlydetected by real time quantitative PCR on biopsies (preferentiallymuscle biopsies, because of the richness in mitochondria in this tissueand the relatively harmless surgical procedure), these tests onlyindicate the average mitochondrial DNA content present in the entiremitochondria population, and this in all the cells contained in thebiopsy, including non-muscle cells present in the biopsy. By contrast,the present invention enables to detect i) alterations in mitochondrialDNA transcription and replication in any single type of cell, includingcells extracted from a buccal sample, which avoids biopsies; ii) theimpairment in mtDNA replication and transcription (which are the outcomeof the mitochondrial DNA molecule) in a portion or in the totality ofmitochondria; iii) the impairment in mtDNA replication and transcriptionin a specific number or in the totality of tested cells. At present,there are indeed no indications whether mitochondrial depletion diseasecells are equally or differently affected in their mitochondrial DNAcontent and activity. Moreover, the present invention enables to revealthe proportion of mitochondria that display at the same timemitochondrial DNA transcription and replication signal, which indicatesefficient cell activity, see FIG. 14 . These data might be of interestor directly useful to follow the progression or even anticipate theprogression of mitochondrial diseases.

The method of the invention, the probes described herein and kitsencompassing said probes or permitting to carry out the method of theinvention can also be used in the analysis and detection of neoplasicdiseases(s) or cancer(s).

The invention provides means useful for the detection and diagnosis ofneoplasic or tumoral cell(s) or tissue(s), and especially to distinguishsaid cell(s) or tissue(s) among healthy cell(s) or tissue(s).

Further experiments have shown a tight association of mt initiation ofDNA replication and mt transcripts in healthy primary cells but not incancer-derived cell lines (FIG. 14 ). In these experiments co-labellingwith mTRANS and mREP probes (purple spots, FIG. 14 )) was performed toreveal the association of mt initiation of DNA replication and mttranscripts. These experiments demonstrated that in HeLa cells about 92%of mitochondrial transcripts were NOT associated with mt DNAreplication), and that in primary fibroblasts only about 27% ofmitochondrial transcripts were NOT associated with mt DNA replication(n=30, from 3 independent experiments). These results were confirmed inother tumor-derived cell lines and primary fibroblasts (data not shown).

Thus, in tested healthy cells mitochondria that are active in DNAreplication were also rich in transcripts (a sign of efficientmitochondrial activity) while in cancer cell lines this occurred only ina small fraction of mitochondria.

The robust activity of mitochondria in healthy cells was confirmed byhigh levels of mitochondrial transcripts (10 to 76-fold higher than inthe cancer-derived cell line, see FIG. 14 b ), a result that is inagreement with the reduced mitochondrial activity in cancer cells (knownas «Warburg effect»).

Thus, the co-labelling of mitochondria with mTRANS and mREP (neverperformed before) measured the efficiency of mitochondrial DNAprocessing in single cells.

The method of the invention could therefore be used as an indicator ofreduced mitochondrial activity, characteristic of cancer cells.

Alterations in mtDNA Processing in Cells with Perturbed mtDNA Content

To assess whether mTRIP detects alterations of DNA processing in cellswith mitochondrial perturbations the inventors examined HeLa rho⁰ cellswhere mtDNA is lacking (Parfait et al. 1998), and HeLa cells treatedwith ethidium bromide (EtBr) for three days to reduce their mtDNAcontent (King and Attardi 1996). Notably, HeLa rho0 cells containedabout one third of the mitochondrial mass (TOM22 immunolabelling)compared to regular HeLa cells, but no signal was detected with eithermTRANS or mREP, confirming the absence of mtDNA transcription andinitiation of replication in these cells (FIG. 16 A). In contrast, cellstreated with EtBr, which had a reduced mtDNA content, maintained aregular mitochondrial mass and displayed a 9.3-fold and a 5.9-foldincrease in the levels of mREP and mTRANS, respectively, compared tountreated cells (FIG. 16 A). High levels of transcripts were confirmedby RT-qPCR of mitochondrial 16S rRNA and CytB. These data indicate thatin spite of mtDNA depletion, EtBr-treated cells dramatically increasedtheir mtDNA replication and transcription activities, likely tocompensate for the low DNA content, in agreement with previous studieson transcription (Seidel-Rogol and Shadel 2002).

Finally, the inventors also examined cells depleted in mtDNA, as is thecase for several diseases (Rotig and Poulton 2009), for example, Rrm2bfibroblasts, carrying a mutation that is associated with a mtDNAdepletion syndrome (Bourdon et al. 2007).

Primary fibroblasts mutated in RRM2B were analyzed. The p53-inducibleribonucleotide reductase subunit which is essential for mtDNA synthesisand is associated with mtDNA depletion syndrome (Bourdon et al. 2007).The inventors found that Rrm2b fibroblasts in spite of a 44% reductionin the mitochondrial mass display a 4-fold reduction in mREP and a3-fold reduction in mTRANS signals compared to normal fibroblasts (FIG.15 B).

Here, reduced mtDNA transcription and replication were observed usingmTRIP. In addition, we noted dramatically increased mitochondrialtranscription and replication signals in cells with depleted mtDNAcontent following treatment with EtBr. This situation likely mimics thenormal amounts of mitochondrial transcripts observed in cells withinduced mtDNA depletion (Seidel-Rogol and Shadel 2002). Moreover, it islikely also representative of cells from patients with a particularlysevere mtDNA depletion, which displayed steady-state levels of mttranscription and had a surprisingly slow progression of the diseasecompared to other mtDNA depletion syndromes (Barthelemy et al. 2001).Thus, mTRIP reveals qualitative and quantitative alterations, whichprovide additional tools for elucidating mitochondrial dysfunction indiseases.

Taken together, the analysis of three different cell types showed thatmREP and mTRANS labelling identify altered or loss of mtDNA processing,which affects mitochondrial function, thus validating mTRIP formonitoring disease states both qualitatively and quantitatively.

Assays on samples of patients diagnosed with mitochondrial diseases havealso been performed. Results are given in FIG. 17 . Conclusions are thatthe reduction of the mitochondrial mass can be associated with eitherreduction or increase of mDNA processing activities. This proves thepotential of the tool for its implementation in myopathies monitoringand/or diagnosis.

Assays on samples of patients diagnosed with diseases not yet known tobe mitochondrial-related were also performed, with the aim to link thedisease to mitochondrial function. Results are given in FIG. 18 .Different mtDNA processing was observed in the “moderate” and “severe”forms of the Cokayne syndrome. As a consequence, mTRIP appears usefulalso for the detection of some diseases which display impairedmitochondrial function (including diseases not known for primarymitochondrial alterations).

The present invention is also of particular interest for testing thecytotoxicity of organic or chemical compounds, especially drugs.

Indeed, the present invention can be used in particular to assay tissuesand organs whose cells are rich in mitochondria, as it is the case forcardiac and skeletal muscle, as well as liver. Therefore the inductionof cytotoxicity by drugs or treatments affecting directly or indirectlythese tissues/organs, can be identified and measured by checkingmitochondrial DNA transcription and replication. Although lethalcytotoxicity can be evaluated with a number of available tests, thepresent invention provides for the detection and quantification ofnon-lethal and transitory cytotoxicity (the one which can have effect onthe long term). To this end, the inventors have shown in HeLa cells thata mild cytotoxic agent (50 μM of H₂O₂—) known to reduce the mtDNAcontent³² results in increase of mREP and mTRANS after a few hours oftreatment, and that these events were associated with increase of themitochondrial mass as well as of the transcription of a mitochondrialbiogenesis factor (FIG. 12B). Since the inventors have used HeLa cells,which are not particularly rich in mitochondria as muscle and livercells are, this experiment indicates that the invention enables thedetection of cytotoxic effects in any type of cells. A more pronounceddetection in mitochondria-rich cells is therefore expected.

H₂O₂ is considered as a low oxidative stress. To check whether mTRIP canbe used to assess mitochondria dysfunction as a cytotoxicity test(preferential use for long term treatments and for products thatprogressively weaken cell function, i.e. anti-inflammatory drugs), thedeveloped mTRIP protocol was also applied to the monitoring of HIVtreatment by AZT. It was demonstrated that mTRIP anticipates thedetection of mitochondrial alterations due to AZT treatment that are notvisible at the level of the mitochondrial function and mitochondrialmass. Results are given in FIG. 19 . Long term treatment with AZT isknown to affect mitochondrial function, in particular in the muscle.With mTRIP, the inventors found that AZT greatly affects mtDNAprocessing in time, in spite of relatively normal values of mtDNAcontent and mt mass. This experiment proves the usefulness of theinvention for use in in vitro detection of progressive myopathiesassociated with antiviral (AZT) HIV treatment. Possibly before theclinical appearance of the myopathy.

Similarly, affected mtDNA processing was demonstrated in cells treatedwith rifampicin (clinical antibiotic). We consider this treatment alsoas a cytotoxic stress. Results are given in FIG. 20 .

These experiments are validating the fact that the invention enables thedetection of mitochondrial impairments due to the use of drugs.

The invention thus also concerns a method for detection of alteredmitochondrial activity in cells comprising the step of detecting thelevel of mitochondrial initation of DNA replication with a first probeof the invention and detecting the level of mitochondrial transcriptswith a second probe of the invention.

The method may be used to detect at the level of single cellsespecially, cells having impaired activity such as cancer cells.

Accordingly, assays on samples of patients diagnosed with cancers werealso performed. Results are given in FIG. 21 . Detection of thelabelling in specific normal and cancer cells (blood cancer cells andsmears of solid cancer cells) was performed, allowing to conclude todifferent levels of mtDNA processing in cancer vs normal cells. Theseexperiments have also allowed concluding to mitochondrial-dependent ATPproduction in cancer cell lines vs normal cells, demonstrating theusefulness of the developed tool for this purpose.

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The invention claimed is:
 1. A nucleic acid molecule that consists ofthe sequence of SEQ ID NO:1, wherein said nucleic acid molecule isdirectly labelled with a fluorescent group.
 2. The nucleic acid moleculeof claim 1, wherein the fluorescent group is fluorescein.
 3. The nucleicacid molecule of claim 1, wherein the fluorescent group is Texas Red. 4.The nucleic acid molecule of claim 1, wherein the fluorescent group isrhodamine.